Nitrophenyl mustard and nitrophenylaziridine alcohols and their corresponding phosphates and their use as targeted cytotoxic agents

ABSTRACT

The present invention relates to a novel nitrophenyl mustard and nitrophenylaziridine alcohols, to their corresponding phosphates, to their use as targeted cytotoxic agents; as bioreductive drugs in hypoxic tumors, and to their use in cell ablation, including gene-directed enzyme-prodrug therapy (GPEPT) and antibody-directed enzymeprodrug therapy (ADEPT), in conjunction with nitroreductase enzymes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of International ApplicationPCT/NZ2004/000275, filed 29 Oct. 2004, which designated the U.S.PCT/NZ2004/000275 claims priority to New Zealand Application Nos. 529249filed 31 Oct. 2003 and 535618 filed 28 Sep. 2004. The entire content ofthese applications are incorporated herein by reference.

The present invention relates to novel nitrophenyl mustard andnitrophenylaziridine alcohols, to their corresponding phosphates, totheir use as targeted cytotoxic agents; as bioreductive drugs in hypoxictumours, and to their use in cell ablation, including gene-directedenzyme-prodrug therapy (GDEPT) and antibody-directed enzyme-prodrugtherapy (ADEPT), in conjunction with nitroreductase enzymes.

BACKGROUND TO THE INVENTION

The use of tumour-selective prodrugs (relatively inactive compounds thatcan be selectively converted to more active compounds in vivo) is avaluable concept in cancer therapy (see, for example Denny, Eur. J. MedChem. (2001) 36, 577).

For example a prodrug may be converted into an anti-tumour agent underthe influence of an enzyme that is linkable to a monoclonal antibodythat will bind to a tumour associated antigen. The combination of such aprodrug with such an enzyme monoclonal/antibody conjugate represents avery powerful clinical agent. This approach to cancer therapy, oftenreferred to as “antibody directed enzyme/prodrug therapy” (ADEPT), isdisclosed in WO88/07378

A further therapeutic approach termed “virus-directed enzyme prodrugtherapy” (VDEPT) has been proposed as a method for treating tumour cellsin patients using prodrugs. Tumour cells are targeted with a viralvector carrying a gene encoding an enzyme capable of activating aprodrug. The gene may be transcriptionally regulated by tissue specificpromoter or enhancer sequences. The viral vector enters tumour cells andexpresses the enzyme, in order that a prodrug is converted to an activedrug within the tumour cells (Huber et al., Proc. Natl. Acad. Sci. USA(1991) 88, 8039). Alternatively, non-viral methods for the delivery ofgenes have been used. Such methods include calcium phosphateco-precipitation, microinjection, liposomes, direct DNA uptake, andreceptor-mediated DNA transfer. These are reviewed in Morgan & French,Annu. Rev. Biochem., 1993, 62; 191. The term “GDEPT” (gene-directedenzyme prodrug therapy) is used to include both viral and non-viraldelivery systems (Denny et al U.S. Pat. No. 6,310,237).

4-Nitroaromatic compounds are reduced by both mammalian and bacterialflavoprotein enzymes, which effect stepwise addition of up to sixelectrons. The major enzymatic metabolite is usually the 4-electronreduced species (hydroxylamine).

A number of nitrophenyl mustards and nitrophenylaziridines have beenreported as prodrugs for use in gene-directed enzyme-prodrug therapy(GDEPT) in conjunction with nitroreductase enzymes. In particular, CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide; (1) [shown below] isreported to be a substrate for the aerobic nitroreductase NTR (nfsB geneproduct) isolated from E. coli B (Boland et al., Biochem. Pharmacol.1991, 41, 867-875; Anlezark et al., Biochem. Pharmacol, 1992, 44,2289-2295; Parkinson et al., J. Med. Chem. 2000, 43, 3624). Thiscompound has been used as a prodrug in both ADEPT (Knox et al., Biochem.Pharmacol., 1995, 49, 1641-1647) and GDEPT (Bridgewater et al.,. Eur. J.Cancer, 1995, 31A, 2362-2370; Bailey et al., Gene Ther., 1996, 3,1143-1150; Bailey and Hart, Gene Ther., 1997, 4, 80-81; Green et al.,Cancer Gene Ther., 1997,4, 229-238) applications, including a clinicaltrial (Chung-Faye et al., Clin. Cancer Res., 2001, 7, 2662-2668).

Similarly, the dinitrophenyl mustard SN 23862 (2) is also a substratefor NTR, and shows selective toxicity towards cell lines that expressthe enzyme. It is activated by nitro group reduction (Palmer et al., J.Med. Chem., 1995, 38, 1229; Kestell et al., Cancer Chemother.Pharmacol., 2000, 46, 365-374). The 4-SO₂Me derivative (3) was also asubstrate (Atwell et al., Anti-Cancer Drug Des., 1996, 11, 553), as werethe regioisomers (4) and (5) (Friedlos et al., J. Med. Chem., 1997, 40,1270).

However, compounds of this type were not very effective as bioreductiveprodrugs when these compounds were activated in hypoxic tumour tissue byendogenous reductase enzymes, showing potency ratios of 2-5 fold underhypoxic conditions relative to oxic conditions in the wild-type AA8 cellline, using a clonogenic assay (Palmer et al., J. Med. Chem. 1996, 39,2518-2528).

Some phosphate analogues of mustards have been described, for thepurpose of solubilising the compounds. The best known is estramustinephosphate (Estracyt; 6), which has been shown to bind to tubulin bindingdomains on various microtubule-associated proteins (Moraga et al.,Biochim. Biophys. Acta, 1992, 1121, 97-103), and which has been shown tobe active in advanced breast cancer (Keren-Rosenberg et al., Semin.Oncol., 1997, 24 (Suppl. 3), 26-29), but has not been shown to beactivated by NTR or hypoxia. Another study has also shown estramustinephosphate to be a radiation sensitizer (Kim et al., Int. J. Radiat.Oncol. Biol. Phys., 1994, 29, 555-557). The phenol mustard phosphateanalogue 7 is a carboxypeptidase substrate that is not activated underhypoxic conditions, (Matsui et al., Japanese Patent 07082280 A2, 1995),and the solubilised mustard 8 has been described as a phosphataseinhibitor but has not been shown to be activated under hypoxicconditions (Workman, Chem.-Biol. Interact., 1978, 20, 103-112).

It is an object of the present invention to provide a specific class ofnitrophenyl mustards and aziridines, bearing short-chain alcohols, andtheir corresponding phosphates for use as targeted cytotoxic agents orbioreductive prodrugs or to at least provide the public with a usefulalternative.

SUMMARY AND DETAILED DESCRIPTION

In a first aspect, the present invention provides novel phosphatecompounds of Formula I

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂or —N(CH₂CHMeW)₂, where each W is independently selected from halogen or—OSO₂Me.Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me; and pharmaceutically acceptable salts and derivatives thereof.

In a preferred embodiment, the phosphate compound of Formula (I) isselected from a compound represented by formulae (Ia), (Ib) or (Ic)

wherein Y may represent

and whereinn represents1 to 6Z represents —NO₂, -halogen, —CN, —CF₃ or —SO₂Me; andwhere each W is independently selected from halogen or —OSO₂Me andpharmaceutically acceptable salts and derivatives thereof.

Preferably, the phosphate compound of Formula (I) is selected from thefollowing:

2-[[2-[Bis(2-bromoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogenphosphate;

3-[[5-[Bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl]amino]propyldihydrogen phosphate;

3-[[5-[Bis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyldihydrogen phosphate;

2-[[2-[Bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyldihydrogen phosphate;

2-[(2-Chloroethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;

2-( {2-[Bis(2-bromopropyl)amino]-3,5-dinitrobenzoyl}amino)ethyldihydrogen phosphate;

2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;

2-[[2- [Bis(2-iodoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogenphosphate;

2-[(2-Iodoethyl)-2,4-dinitro-6-({[2-(phosphonooxy)ethyl]amino}carbonyl)-anilino]ethyl methanesulfonate;

2-[(2-Chloroethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate;

3-({3-[Bis(2-bromoethyl)amino]-2,6-dinitrobenzoyl}amino)propyldihydrogen phosphate;

2-[(2-Bromoethyl)-2,4-dinitro-3-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;

2-[(2-Bromoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate; and

2-[(2-Iodoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate.

In a second aspect, the present invention provides alcohol compounds ofFormula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂,or—N(CH₂CH MeW)₂ where each W is independently selected from halogen or—OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof, with the proviso that

are excluded.

In a preferred embodiment, the alcohol compound of Formula (II) isselected from a compound represented by formulae (IIa), (IIb) or (IIc)

wherein Y may represent

and whereinn represents1 to 6Z represents —NO₂, -halogen, —CN, —CF₃ or —SO₂Me; andwhere each W is independently selected from halogen or —OSO₂Me andpharmaceutically acceptable salts and derivatives thereof with theproviso that

are excluded.

Preferably the compound of Formula (H) is selected from the following:

N-(3-Hydroxypropyl)-5-[bis(2-chloroethyl)amino]-2,4-dinitrobenzamide;

N-(3-Hydroxypropyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide;

N-(2-Hydroxyethyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide;

N-(4-Hydroxybutyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide;

N-(5-Hydroxypentyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide;

N-(6-Hydroxyhexyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide;

5-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide;

2[(2-Bromoethyl)-5-[[(3-hydroxypropyl)amino]carbonyl]-2,4-dinitroanilino]ethylmethanesulfonate;

5-[Bis(2-iodoethyl)amino]-N-(2-hydroxyethyl)-2,4-dinitrobenzamide;

2-[Bis(2-Chloroethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide;

2-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide;

2-[Bis(2-chloroethyl)amino]-N-(3-hydroxypropyl)-3,5-dinitrobenzamide;

2-[Bis(2-bromoethyl)amino]-N-(3-hydroxypropyl)-3,5-dinitrobenzamide;

2-[Bis(2-chloroethyl)amino]-N-(4-hydroxybutyl)-3,5-dinitrobenzamide;

2-[Bis(2-bromoethyl)amino]-N-(4-hydroxybutyl)-3,5-dinitrobenzamide;

2-[Bis(2-chloroethyl)amino]-N-(5-hydroxypentyl)-3,5-dinitrobenzamide;

2-[Bis(2-bromoethyl)amino]-N-(5-hydroxypentyl)-3,5-dinitrobenzamide;

2-[Bis(2-chloroethyl)amino]-N-(6-hydroxyhexyl)-3,5-dinitrobenzamide;

2-[Bis(2-bromoethyl)amino]-N-(6-hydroxyhexyl)-3,5-dinitrobenzamide;

2-[Bis(2-bromopropyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide;

2-((2-Bromoethyl)-2-{[(2-hydroxypropyl)amino]carbonyl}-4,6-dinitroanilino)ethylmethanesulfonate;

2-((2-Bromoethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethylmethanesulfonate;

2-((2-Chloroethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethylmethanesulfonate;

2-[Bis(2-iodoethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide;

2-((2-Iodoethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethylmethanesulfonate;

3-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-2,6-dinitrobenzamide;

2-((2-Bromoethyl)-3-{[(2-hydroxyethyl)amino]carbonyl}-2,4-dinitroanilino)ethylmethanesulfonate;

3-[Bis(2-bromoethyl)amino]-N-(3-hydroxypropyl)-2,6-dinitrobenzamide;

2-((2-bromoethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethylmethanesulfonate;

3-[Bis(2-bromoethyl)amino]-N-(4-hydroxybutyl)-2,6-dinitrobenzamide;

2-((2-Bromoethyl)-3-{[(4-hydroxybutyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate;

2-((2-Chloroethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethylmethanesulfonate; and

2-((2-Iodoethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate.

In a third aspect of the invention there is provided a method ofpreparing the phosphates is represented by the general formula (I);

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom;Y represents at any available ring position -N-aziridinyl or—N(CH₂CH₂W)₂, where each W is independently selected from halogen or—OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;and pharmaceutically acceptable salts and derivatives thereof; themethod including the step of

-   -   (i) phosphorylating a compound of formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂,or —N(CH₂CH MeW)₂ where each W is independently selected from halogen or—OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me; andR represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom.

In a fourth aspect there is provided a method of preparing a compound offormulae (IIa), (IIb) or (IIc)

wherein Y may represent

and whereinn represents1 to 6Z represents —NO₂, -halogen, —CN, —CF₃ or —SO₂Me; andwhere W₁ is halogen and W₂ is —OSO₂Me and pharmaceutically acceptablesalts and derivatives thereof;the method including the step of reacting a compound of formulae (IIa′),(IIb′) or (IIc′) optionally with heating

wherein Y may represent

wherein W′₁ and W′₂ are each halogen;with an effective amount of silver methanesulfonate (AgOMs) in a solventto give a compound of formulae (IIa), (IIb) or (IIc) defined above.

It is to be appreciated that in the method defined immediately abovewhere W′₁ and W′₂ are either iodine and/or bromine that the iodineand/or bromine can be partially or completely substituted with —OSO₂Me.In the situation where either or both of W′₁ and W′₂ represent chlorine,the chlorine is inert and cannot be can be substituted with —OSO₂Me.

Preferably the solvent is selected from MeCN or other polar non-proticsolvent.

In a fifth aspect there is provided a method of preparing a compound offormulae (Ia), (Ib) or (Ic)

wherein Y may represent

and whereinn represents1 to 6Z represents —NO₂, -halogen, —CN, —CF₃ or —SO₂Me; andwhere each W is independently selected from halogen or —OSO₂Me andpharmaceutically acceptable salts and derivatives thereofthe method including the step of phosphorylating a compound representedby formulae (IIa), (IIb) or (IIc)

wherein Y represents

and whereinn represents1 to 6Z represents —NO₂, -halogen, —CN, —CF₃ or —SO₂Me; andwhere each W is independently selected from halogen or —OSO₂Me andpharmaceutically acceptable salts and derivatives.

In a sixth aspect there is provided a compound of formula (I), formula(Ia), (Ib) or (Ic) or formula (IIa), (IIb) or (IIc) obtained by any oneof the preparative methods defined above.

In a seventh aspect, the present invention provides a method for the useas prodrugs suitable for

-   -   (i) GDEPT (gene-directed enzyme-prodrug therapy) or ADEPT        (anti-body directed enzyme prodrug therapy) in conjunction with        at least one nitroreductase enzyme; or    -   (ii) Use as one or more hypoxia-selective cytotoxins,        including the step of administering a compound of Formula (I) as        defined above or a compound of Formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂,or —N(CH₂CH MeW)₂ where each W is independently selected from halogen or—OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof;or a mixture thereof in a therapeutically effective amount to tumourcells in a subject.

Preferably, the nitroreductase enzyme is encoded for by the nfsB gene ofeither E. coli or by orthologous genes in Clostridia species.

Preferably the method includes the further step of irradiating thetumour cells.

In an eighth aspect, the present invention provides a method for the useas prodrugs suitable for GDEPT (gene-directed enzyme-prodrug therapy) orADEPT (antibody-directed enzyme prodrug therapy) in conjunction with atleast one nitroreductase enzyme, as an anticancer agent including thestep of administering a compound of Formula (I) as defined above or acompound of Formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂or —N(CH₂CH MeW)₂, where each W is independently selected from halogenor —OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof;or a mixture thereof in a therapeutically effective amount to targettumour cells in a subject.

Preferably the nitroreductase enzyme is encoded for by the nfsB gene ofE. coli or by orthologous genes in Clostridia species.

Preferably the method includes the further step of irradiating thetumour cells.

It is to be appreciated that with ADEPT it may be necessary to supply areducing co-factor, because these may not be present in significantconcentrations outside cells. It is envisaged that a synthetic co-factorcould be used to stimulate activation of the pro-drug by the likes of anintracellular enzyme. The same issue does not arise with GDEPT becausethere are several intracellular reducing co-factors such as the likes ofNADH and NADPH in significant concentrations.

In a ninth aspect of the present invention, there is provided a methodof cell ablation utilising at least one nitroreductase enzyme, whereinthe method includes the step of administering a compound of Formula (I)as defined a above or a compound of Formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂or —N(CH₂CH MeW)₂, where each W is independently selected from halogenor —OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof, or a mixture thereof in an effective amount toablate cells, wherein said cells express at least one nitroreductaseenzyme.

Preferably the nitroreductase enzyme is encoded for by the nfsB gene inE. coli or by orthologous genes in Clostridia species.

Preferably, the cells that are targeted for ablation are tumor cells intissue in a subject.

Preferably, the method of cell ablation utilising at least onenitroreductase enzyme is delivered by either ADEPT or GDEPT technology.

Preferably, the cell ablation provides a substantially minimal bystandereffect.

In a tenth aspect, the present invention provides a method of providinganticancer therapy, wherein a compound of Formula (D as defined above isadministered in a therapeutically effective amount to tumour cells in asubject.

Preferably the therapeutically effective amount of said method isbetween about 20% to 100% of the maximum tolerated dose of said subject.

Preferably, the method includes the further step of irradiating thetumour cells.

In an eleventh aspect of the present invention there is provided apharmaceutical composition including a therapeutically effective amountof a compound of Formula (I) or a compound of Formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂or —N(CH₂CH MeW)₂, where each W is independently selected from halogenor —OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof, or a mixture thereof, and a pharmaceuticallyacceptable excipient, adjuvant, carrier, buffer or stabiliser.

The pharmaceutically acceptable excipient, adjuvant, carrier, buffer orstabiliser should preferably be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, such as cutaneous, subcutaneous, orintravenous. It is to be appreciated that these factors could be readilydetermined by someone skilled in the art without undue experimentation.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvent. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such as gelatin.

For intravenous, cutaneous or subcutaneous injection, the activeingredient will be in the form of a parenterally acceptable aqueoussolution which is pyrogen-free and has a suitable pH, isotonicity andstability. Those of relevent skill in the art are well able to preparesuitable solutions using, for example, isotonic vehicles such as SodiumChloride injection, Ringer's injection, Lactated Ringer's injection.Preservatives, stabilisers, buffers antioxidants and/or other additivesmay be included as required.

In a twelfth aspect of the present invention there is provided, the usein the manufacture of a medicament of an effective amount of a compoundof Formula (I) as defined above or a compound of Formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂or —N(CH₂CH MeW)₂, where each W is independently selected from halogenor —OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁- alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof, or mixtures thereof, for use in (i) GDEPT or (ii)as a hypoxia selective cytotoxin, to target cancer cells in a subject inneed thereof.

In a thirteenth aspect of the present invention there is provided, theuse in the manufacture of a medicament of an effective amount of acompound of Formula (I) as defined above or a compound of Formula (II)

wherein:X represents at any available ring position —CONH—, —SO₂NH—, —O—, —CH₂—,—NHCO— or —NHSO₂—;Y represents at any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂or —N(CH₂CH MeW)₂, where each W is independently selected from halogenor —OSO₂Me;Z represents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me;R represents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom; and pharmaceutically acceptable salts andderivatives thereof, or mixtures thereof for use in cell ablationtherapy to target cancer cells in a subject in need thereof.

While the compounds of the present invention will typically be used totarget tumour cells or tumour tissues in human subjects, they may beused to target tumour cells or tissues in other warm blooded animalsubjects such as other primates, farm animals such as cattle, and sportsanimals and pets such as horses, dogs, and cats.

As used throughout the specification the term “therapeutically effectiveamount”, is to be understood as an amount of a compound of Formula (I)or Formula (II) as defined above or a compound of any one of compoundsIa-Ic, or IIa-IIc as defined above or a mixture thereof that issufficient to show benefit to a subject with cancer cells. The actualamount, rate and time-course of administration, will depend on thenature and severity of the disease being treated. Prescription oftreatment is within the responsibility of general practitioners andother medical doctors.

As used throughout the specification the term “halogen” includeschlorine, bromine or iodine.

It is to be understood that the compounds of the invention as definedabove may be administered alone or in combination with other treatments,especially radiotherapy and cytotoxic chemotherapeutic drugs, eithersimultaneously or sequentially dependent upon the condition to betreated.

As used throughout the specification the pharmaceutically acceptablederivatives and salts thereof include acid derived salts formed from arehydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic,salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic,isethionic acids and the like and base derived salts formed from sodiumand potassium carbonate, sodium and potassium hydroxide, ammonia,triethylamine, triethanolamine and the like.

As used throughout the specification, the term cell ablation is to beunderstood as the killing of cells, that have been engineered to expressan enzyme, such as a nitroreductase, by administration of a prodrug thatis activated by that enzyme. As a result, cell ablation can be used toselectively ablate specified target cells or tissue through specificenzymatic expression of a nitroreductase for example, that isspecifically expressed by the tissue and which can then be employed toactivate a prodrug into an active metabolite to ablate the specifiedtarget cells or tissue. (Gusterson et al. Endocrine Related Cancer,1997, 4, 67-74.)

The expression “substantially minimal bystander effect” is to beunderstood as meaning that the killing of adjoining non-targeted cellsis minimal because there is little or no diffusion between the targetedand non-targeted cells of an activated metabolite that arises from theenzymatic activation of a compound of Formula (I) or Formula (II) asdefined above or a compound of any one of compounds Ia-Ic, or IIa-IIc asdefined above or a mixture thereof.

Pharmaceutically acceptable salts of formula (I) include the basic oracidic compounds of formula (I) that form pharmaceutically acceptablesalts with both organic and inorganic acids and/or organic and inorganicbases. Examples of suitable acids for salt formation are hydrochloric,sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,fumaric, succinic, ascorbic, maleic, methanesulfonic, isethionic, andthe like. Examples of suitable bases for salt formation are sodium andpotassium carbonate, sodium and potassium hydroxide, ammonia,triethylamine, triethanolamine, and the like.

Further aspects of the present invention will become apparent from thefollowing description given by way of example only and with reference tothe accompanying synthetic schemes.

Examples of the compounds of Formula (I) where X is —CONH— can beprepared by the processes described in Scheme 1, where Z is as definedabove for Formula (I).

The following Tables 1a and 2a set out physical data for compoundswithin the general Formula (I) and (II), representative of it, andcapable of being prepared by the processes of the invention.

wherein Y may represent

Table 1a. Representative examples of parent alcohols

TABLE 1a Representative examples of parent alcohols Y No Z Y (W₁, W₂) nmp formula or ref analyses IIa-1 NO₂ aziridines — 2 192–193 Ref. 1 C, H,N IIa-2 NO₂ — Cl, Cl 2 Ref. 2 IIa-3 NO₂ — Cl, Cl 3 90–91 Ref 4 C, H, N,Cl IIa-7 NO₂ — Br, Br 2 151–152 C₁₃H₁₆Br₂N₄O₆ C, H, N, Br IIa-7s SO₂Me —Br, Br 2 126–127 C₁₄H₁₉Br₂N₃O₆S C, H, N IIa-8 NO₂ — Br, Br 3 85–86 Ref 4C, H, N, Br IIa-9 NO₂ — Br, Br 4 123–124 C₁₅H₂₀Br₂N₄O₆ C, H, N, BrIIa-10 NO₂ — Br, Br 5 gum C₁₆H₂₂Br₂N₄O₆ HRMS IIa-11 NO₂ — Br, Br 6 gumC₁₇H₂₄Br₂N₄O₆ HRMS IIa-12 NO₂ — Br, OMs 2 Ref. 2 IIa-13 NO₂ — Br, OMs 3gum C₁₆H₂₁BrN₄O₉S HRMS IIa 14 NO₂ — I, I 2 142–143 C₁₃H₁₆I₂N₄O₆ C, H, N,I IIb-1 — aziridines — 6 189–192 C₁₅H₂₀N₄O₆ C, H, N IIb-2 — — Cl, Cl 2109–111 C₁₃H₁₆Cl₂N₄O₆ C, H, N IIb-3 — — Cl, Cl 3 89–91 C₁₄H₁₈Cl₂N₄O₆ C,H, N, Cl IIb-4 — — Cl, Cl 4 gum C₁₅H₂₀Cl₂N₄O₆ HRMS IIb-5 — — Cl, Cl 5gum C₁₆H₂₂Cl₂N₄O₆ HRMS IIb-6 — — Cl, Cl 6 gum C₁₇H₂₄Cl₂N₄O₆ HRMS IIb-2mCl, OMs 2 gum C₁₄H₉ClN₄O₉S HRMS IIb-7 — — Br, Br 2 105–108 C₁₃H₁₆Br₂N₄O₆C, H, N, Br IIb-7a — — Br, Br^(A) 2 127–130 C₁₅H₂₀Br₂N₄O₆ C, H, N IIb-8— — Br, Br 3 89–94 C₁₄H₁₈Br₂N₄O₆ C, H, N, Br IIb-9 — — Br, Br 4 gumC₁₅H₂₀Br₂N₄O₆ HRMS IIb-10 — — Br, Br 5 gum C₁₆H₂₂Br₂N₄O₆ HRMS IIb-11 — —Br, Br 6 gum C₁₇H₂₄Br₂N₄O₆ HRMS IIb-12 — — Br, OMs 2 Ref. 3 IIb-13 — —Br, OMs 3 gum C₁₅H₂₁BrN₄O₉S HRMS IIb-14 — — I, I 2 129–131 C₁₃H₁₆I₂N₄O₆C, H, N IIb-15 — — I, OMs 2 gum C₁₄H₁₉IN₄O₉S HRMS IIc-6 Cl, OMs 3104–109 C₁₅H₂₁ClN₄O₉S C, H, N IIc-7 — — Br, Br 2 gum C₁₃H₁₆Br₂N₄O₆ HRMSIIc-8 — — Br, Br 3 gum C₁₄H₁₈Br₂N₄O₆ HRMS IIc-9 — — Br, Br 4 gumC₁₅H₂₀Br₂N₄O₆ HRMS IIc-12 — — Br, OMs 2 94–97 C₁₄H₁₉BrN₄O₉S C, H, NIIc-13 — — Br, OMs 3 115–117 Ref. 3 C, H, N IIc-14 — — Br, OMs 4 114–117C₁₆H₂₃BrN₄O₉S C, H, N IIc-15 I, OMs 3 100–103 C₁₅H₂₁IN₄O₉S C, H, N^(A)α-methyl mustard Notes References for known compounds. 1. Khan AH,Ross WCJ. Tumor-growth inhibitory nitrophenylaziridines and relatedcompounds. Structure-activity relations. II. Chem.-Biol. Int., 1971, 4,11–22. 2 NZ Patent No. 240785 3. Co-pending NZ Application No. 521851 4.Wilson WR, Pullen SM, Hogg A, Helsby NA, Hicks KO, Denny WA.Quantitation of bystander effects in nitroreductase suicide gene therapyusing three-dimentional cell cultures. Cancer Res., 2002, 62, 1425–1432.

The compounds of Table 1a can be prepared by the general methods set outin Schemes 2a-2k, and exemplified in Examples 1-20 below.

where Y may represent

TABLE 1b Examples of phosphates of formulae Ia–Ic Y (W₁, No Z W₂) n mpformula analyses Ia-3P NO₂ Cl, Cl 3 195–200 C₁₄H₁₉Cl₂N₄O₉P HRMS Ia-8PNO₂ Br, Br 3 170–174 C₁₄H₁₉Br₂N₄O₉P HRMS Ib-2P — Cl, Cl 2 FoamC₁₃H₁₇Cl₂N₄O₉P HRMS Ib-2mP Cl, 2 132–134 C₁₄H₂₀ClN₄O₁₂PS C, H, N OMsIb-7P — Br, Br 2 Foam C₁₃H₁₇Br₂N₄O₉P HRMS Ib-7aP — Br, Br^(A) 2 157–161C₁₅H₂₁Br₂N₄O₉P C, H, N Ib-12P — Br, 2 Foam C₁₄H₂₀BrN₄O₁₂PS HRMS OMsIb-14P — I, I 2 Foam C₁₃H₁₇I₂N₄O₉P HRMS Ib-15P — I, OMs 2 147–150C₁₄H₂₀IN₄O₁₂PS C, H, N Ic-6P Cl, 3 88–92 C₁₅H₂₂ClN₄O₁₂PS C, H, N OMsIc-8P — Br, Br 3 Foam C₁₄H₁₉Br₂N₄O₉P HRMS Ic-12P Br, 2 93–97C₁₄H₂₀BrN₄O₁₂PS C, H, N OMs Ic-13P — Br, 3 foam C₁₅H₂₂BrN₄O₁₂PS HRMS OMsIc-15P I, OMs 3 C₁₅H₂₂IN₄O₁₂PS ^(A)alpha-Me

The compounds of Table 1b can be prepared by the general methods set outin Scheme 3, and exemplified in Examples 26-39 below.

In Scheme 3, X, Y, Z, and R are as specified for formula (I) and (II)above.

EXAMPLES

The invention and the best mode for practising the same are illustratedby the following Examples 1-25 (alcohols) and Examples 26-39(phosphates).

Example 1 (Scheme 2a)N-(3-Hydroxypropyl)-5-[bis(2-Chloroethyl)Amino]-2,4-Dinitrobenzamide(IIa-3)

A suspension of 5-[bis(2-chloroethyl)amino]-2,4-dinitrobenzoic acid[Palmer et al., J. Med. Chem., 1994, 37, 2175] (1) (2.50 g, 7.1 mmol) inSOCl₂ (20 mL) containing DMF (2 drops) was heated under reflux for1 h,then concentrated to dryness under reduced pressure and re-evaporatedwith benzene. The resulting crude benzoyl chloride was dissolved inMe₂CO (50 mL) and the cooled (−5° C.) solution was treated with a coldsolution of 3-amino-1-propanol (1.09 g, 14.5 mmol) in water (25 mL). Thereaction mixture was shaken at room temperature for 5 min, then dilutedwith water (25 mL), concentrated to half volume, and extracted withCH₂Cl₂ (2×). The organic extract was washed with 0.1 N HCl and waterthen worked up to give a solid which was chromatographed on silica gel,eluting with EtOAc to give IIa-3 (2.37 g, 82%): mp (EtOAc/i-Pr₂O) 90-91°C.; ¹H [(CD₃)₂SO] δ8.63 (t, J=5.6 Hz, 1 H, CONH), 8.53 (s, 1 H, H-3),7.42 (s, 1 H, H-6), 4.46 (t, J=5.1 Hz, 1 H, OH), 3.82 (t, J=5.9 Hz, 4 H,N(CH₂CH₂Cl)₂), 3.68 (t, J=5.9 Hz, 4 H, N(CH₂CH₂Cl)₂), 3.49 (q, J=6.0 Hz,2 H, CH₂OH), 3.29 (q, partially obscured, J=5.9 Hz, 2 H, CONHCH₂), 1.68(pent, J=6.7 Hz, 2 H, CH₂CH₂CH₂). Anal. (C₁₄H₁₈Cl₂N₄O₆) C, H, N, Cl.

Example 2 (Scheme 2a)N-(3-Hydroxypropyl)-5-[Bis(2-Bromoethyl)Amino]-2,4-Dinitrobenzamide(IIa-8)

A suspension of powdered 5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzoicacid (2) (1.10 g, 2.49 mmol) in benzene (170 mL) was treated at 20° C.with oxalyl bromide (1.10 mL, 11.7 mmol) and DMF (2 drops). The mixturewas stirred at 20° C. for 2 h, then concentrated under reduced pressure,and re-evaporated to dryness in the presence of benzene under highvacuum. The resulting acid bromide was dissolved in Me₂CO (20 mL) andthe solution was treated at −5° C. with a cold solution of3-amino-1-propanol (0.39 g, 5.19 mmol) in water (10 mL). The mixture wasshaken at room temperature for 5 min, then diluted with water andextracted with EtOAc (2×). The organic extract was worked up and theresulting residue was chromatographed on silica gel, eluting with EtOAc,to give IIa-8 (1.06 g, 85%): mp (EtOAc/i-Pr₂O) 85-86° C.; ¹H NMR[(CD₃)₂SO] δ 8.64 (t, J=5.6 Hz, 1 H, CONH), 8.53 (s, 1 H, H-3), 7.41 (s,1 H, H-6), 3.77-3.64 (m, 8 H, N(CH₂CH₂Cl)₂), 4.46 (br s, 1 H, OH), 3.49(t, J=6.3 Hz, 2 H, CH₂OH), 3.33-3.25 (m, partially obscured, 2 H,CONHCH₂), 1.68 (pent, J=6.72 Hz, 2 H, CH₂CH₂CH₂). Anal. (C₁₄Hl₁₈Br₂N₄O₆)C, H, N, Br.

Example 3 (Scheme 2a)N-(2-Hydroxyethyl)-5-[Bis(2-Bromoethyl)Amino]-2,4-Dinitrobenzamide(IIa-7)

Similar reaction of the acid bromide of 2 with 2-aminoethanol gave IIa-7(0.78 g, 46%): mp (MeOH/EtOAc/pet. ether) 151-152° C.; ¹H NMR [(CD₃)₂SO]δ 8.73 (t, J=5.7 Hz, 1 H, CONH), 8.53 (s, 1 H, H-3), 7.43 (s, 1 H, H-6),4.76 (t, J=5.6 Hz, 1 H, OH), 3.77-3.64 (m, 8 H, N(CH₂CH₂Br)₂), 3.53 (q,J=6.0 Hz, 2 H, CH₂OH), 3.31 (q, partially obscured, J=6.1 Hz, 2 H,CONHCH₂). Anal. (C₁₃H₁₆Br₂N₄O₆) C, H, N, Br.

Example 4 (Scheme 2a)N-(4-Hydroxybutyl)-5-[Bis(2-Bromoethyl)Amino]-2,4-Dinitrobenzamide (IIa-9)

Similar reaction of the acid bromide of 2 with 4-amino-1-butanol in coldMe₂CO, followed by chromatography on silica gel and elution with EtOAcgave IIa-9 (69%) as a yellow solid: mp (EtOAc/iPr₂O) 123-124° C.; ¹H NMR[(CD₃)₂SO] δ 8.62 (t, J=5.6 Hz, 1 H), 8.53 (s, 1 H), 7.39 (s, 1 H), 4.39(t, J=5.1 Hz, 1 H), 3.78-3.64 (m, 8 H), 3.47-3.40 (m, 2 H), 3.27-3.20(m, 2 H), 1,61-1.44 (m, 4 H). Anal. (C₁₅H₂₀Br₂N₄O₆) C, H, N, Br.

Example 5 (Scheme 2a)N-(5-Hydroxypentyl)-5-[Bis(2-Bromoethyl)Amino]-2,4-Dinitrobenzamide(IIa-10)

Similar reaction of the acid bromide of 2 with 5-amino-1-pentanol incold Me₂CO, followed by chromatography on silica gel and elution withEtOAc gave IIa-10 (66%) as a yellow foam; ¹H [(CD₃)₂SO] δ 8.62 (t, J=5.6Hz, 1 H), 8.53 (s, 1 H), 7.38 (s, 1 H), 4.34 (t, J=5.1 Hz, 1 H),3.79-3.64 (m, 8 H), 3.44-3.37 (m, 2 H), 3.26-3.18 (m, 2 H), 1.59-1.29(m, 4 H). HRMS (FAB) Calcd. for C₁₆H₂₃ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 524.9984,found 524.9964.

Example 6 (Scheme 2a)N-(6-Hydroxyhexyl)-5-[Bis(2-Bromoethyl)Amino]-2,4-Dinitrobenzamide(IIa-10)

Similar reaction of the acid bromide of 2 with 6-amino-1-hexanol in coldMe₂CO, followed by chromatography on silica gel and elution with EtOAcgave IIa-11 (72%) as a yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.61 (t, J=5.6Hz, 1 H), 8.53 (s, 1 H), 7.38 (s, 1 H), 4.31 (t, J=5.2 Hz, 1 H),3.79-3.64 (m, 8 H), 3.43-3.36 (m, 2 H), 3.27-3.19 (m, 2 H), 1.58-1.26(m, 4 H). HRMS (FAB) Calcd. for C₁₇H₂₅ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 539.0141,found 539.0137.

Example 7 (Scheme 2b)5-[Bis(2-Bromoethyl)Amino]-N-(2-hydroxyethyl)-4-(Methylsulfonyl)-2-Nitrobenzamide(IIa-7s)

5-Fluoro-4-(methylsulfonyl)-2-nitrobenzoic acid [Atwell et al., ACDD,1996, 11, 553] (3) was heated in excess SOCl₂/catalytic DMF to providethe acid chloride, which was reacted with dry MeOH to give methyl5-fluoro-4-(methylsulfonyl)-2-nitrobenzoate (4): mp (EtOAc/hexane)134-135° C.; ¹H NMR [(CD₃)₂SO] δ 8.49 (d, J=5.9 Hz, 1 H), 8.14 (d, J=9.3Hz, 1 H), 3.92 (s, 3 H), 3.46 (s, 3 H). Anal. (C₉H₈FNO₆S) C, H, N.

A mixture of 4 (1.48 g, 5.34 mmol) and diethanolamine (1.40 g, 13.3mmol) in DMA (6 mL) was stirred at 30° C. for1 h, and then diluted withEtOAc (60 mL). The solution was washed with brine (2×) and concentratedunder reduced pressure. The residue was purified by chromatography onsilica gel, eluting with EtOAc/MeOH, followed by recrystallization fromEtOAc/iPr₂O, to give methyl5-[bis(2-hydroxyethyl)amino]-4-(methylsulfonyl)-2-nitrobenzoate (5)(1.41 g, 73%): mp 99-100° C.; ¹H NMR [(CD₃)₂SO] δ 8.56 (s, 1 H), 7.73(s, 1 H), 4.62 (t, J=4.9 Hz, 2 H), 3.89 (s, 3 H), 3.59-3.49 (m, 8 H),3.45 (s, 3 H). Anal. (C₁₃H₁₈N₂O₈S) C, H, N.

A solution of 5 (1.48 g, 4.08 mmol) in dry pyridine (15 mL) was treateddropwise at 0° C. with MsCl (0.80 mL, 10.3 mmol). The reaction wasstirred at 0° C. for 2 h, then poured into 10% aqueous NaBr. Theresulting crude dimesylate was collected, washed well with water, dried,dissolved in DMF (15 mL) and stirred with NaBr (21.6 g, 25 mmol) at 70°C. for 1.5 h. The cooled mixture was poured into water and the resultingsolid was purified by chromatography, on silica gel, eluting withCH₂Cl₂, then recrystallisation from CH₂Cl₂/iPr₂O to give methyl5-[bis(2-bromoethyl)amino]-4-(methylsulfonyl)-2-nitrobenzoate (6) (1.47g, 74%): mp 161-162° C.; ¹H NMR [(CD₃)₂SO] δ 8.58 (s, 1 H), 7.94 (s, 1H), 3.90 (s, 3 H), 3.82 (t, J=7.0 Hz, 4 H), 3.63 (t, J=6.9 Hz, 4 H),3.48 (s, 3 H). Anal. (C₁₃H₁₆Br₂N₂O₆S) C, H, N.

A solution of 6 (1.00 g, 2.05 mmol) in a dioxane/MeOH (1:1, 20 mL) wastreated at 10° C. with 4N aqueous KOH (5 mL), and stirred at 10° C. for45 min. The mixture was acidified to pH 2 with1 N aqueous HBr,concentrated to a small volume under reduced pressure, and then dilutedwith saturated aqueous NaBr (20 mL). The resulting semi-solid wasisolated and crystallized twice from MeOH/H₂O to give5-[bis(2-bromoethyl)amino]-4-(methylsulfonyl)-2-nitrobenzoic acid (7)(0.70 g, 72%): mp 174-176° C.; ¹H NMR [(CD₃)₂SO] δ 8.50 (s, 1 H), 7.88(s, 1 H), 3.79 (t, J=7.0 Hz, 4 H), 3.62 (t, J=7.0 Hz, 4 H), 3.48 (s, 3H). Anal. (C₁₂H₁₄Br₂N₂O₆S) C, H, N.

A finely-divided suspension of 7 (260 mmg, 0.55 mmol) in dry benzene (50mL) was treated with (COBr)₂ (2.13 mL, 0.20 mmol) and catalytic DMF. Themixture was stirred for 2 h, then concentrated to dryness under reducedpressure and re-evaporated with benzene under high vacuum. The resultingcrude acid bromide was dissolved in Me₂CO (10 mL) and treated at −5° C.with a cold solution of 2-aminoethanol (101 mg, 1.65 mmol) in water (5mL). The mixture was stirred at 0° C. for 5 min, then acidified to pH 4with1 N aqueous HBr, and concentrated under reduced pressure. Theresidue was chromatographed on silica gel, eluting with EtOAc, to giveIIa-7s (222 mg, 78%): mp (EtOAc/iPr₂O) 126-127° C.; ¹H NMR [(CD₃)₂SO] δ8.75 (t, J=5.6 Hz, 1 H), 8.51 (s, 1 h), 7.68 (s, 1 H), 4.79 (t, J=5.4Hz, 1 H), 3.76 (t, J =7.1 Hz, 4 H), 3.62 (t, J=7.0 Hz, 4 H), 3.54 (q, J=5.9 Hz, 2 H), 3.48 (s, 3 H), 3.31 (after D₂O exchange, t, J=6.0 Hz, 2H). HRMS (FAB) calcd. for C₁₄H₂₀ ⁷⁹Br₂N₃O₆S (MH⁺) m/z 515.9440; found515.9425.

Example 8 (Scheme 2c) 2[(2-Bromoethyl)-5-[[(3-Hydroxypropyl)Amino]Carbonyl]-2,4-Dinitroanilino]EthylMethanesulfonate (IIa-13) and5-[Bis(2-Iodoethyl)Amino]-N-(2-Hydroxyethyl)-2, 4-Dinitrobenzamide(IIa-14)

5-(Bis{2-[(methylsulfonyl)oxy]ethyl}amino)-2,4-dinitrobenzoic acid [Amethod of preparing this compound is disclosed in co-pending NZApplication No. 521851] (9) was heated under reflux in excess SOCl₂ (60mL) and catalytic DMF for1 h. Evaporation under reduced pressure,followed by azeotroping in benzene, gave the crude acid chloride. Thiswas dissolved in dry Me₂CO and treated at 0° C. with 3-amino-1-propanolat 0° C. for 5 min. The mixture was acidified to pH 2-3 with 0.2 N HCl,concentrated to half volume, and then solid NaBr was added, followed byextraction with EtOAc (2×). Evaporation, and chromatography of theresidue on silica gel, eluting with EtOAc/MeOH (9:1), gave give2-(5-{[(3-hydroxypropyl)amino]carbonyl }{2-[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethyl methanesulfonate(8) (68%) as a yellow gum; ¹H NMR [(CD₃)₂SO] δ 8.54 (t, J=5.7 Hz, 1 H),8.53 (s, 1 H), 7.45 (s, 1 H), 4.43 (t, J=5.1 Hz, 1 H), 4.33 (t, J=5.2Hz, 4 H), 3.69 (t, J=5.2 Hz, 4 H), 3.57 (q, J=5.9 Hz, 2 H), 3.26 (afterD₂O exchange, t, J=7.0 Hz, 2 H), 3.12 (s, 6 H), 1.66 (pent, J=6.7 Hz, 2H). HRMS (FAB) calcd. for C₁₆H₂₅N₄O₁₂S (MH⁺) m/z 529.0910; found529.0904.

A solution of 8 in DMF was treated with LiBr (1.4 equiv.), and worked upas above, and the product was chromatographed on silica gel. Elutionwith EtOAc gave a small amount of the dibromo mustard, while elutionwith EtOAc/MeOH (19:1) gave IIa-13 (31%) as a yellow gum: ¹H NMR[(CD₃)₂SO] δ 8.60 (t, J=5.6 Hz, 1 H), 8.54 (s, 1 H), 7.44 (s, 1 H), 4.45(t, J=5.2 Hz, 1 H), 4.33 (t, J=5.1 Hz, 2 H), 3.74 (t, J=5.2 Hz, 2 H),3.72-3.66 (m, 4 H), 3.49 (q, J=5.9 Hz, 2 H), 3.27 (after D₂O exchange,t, J=7.0 Hz, 2 H), 3.14 (s, 3 H), 1.68 (pent, J=6.7 Hz, 2 H). HRMS (FAB)calcd. for C₁₅H₂₂ ⁷⁹BrN₄O₉S (MH⁺) m/z 515.0270; found 515.0283.

Similar treatment of the acid chloride of 9 (activation with(COCl)₂/DMF) with 2-aminoethanol gave2-(5-{[(2-hydroxyethyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethyl methanesulfonate(10). A stirred mixture of 10 (1.42 g, 2.76 mmol) and NaI (3.3 g, 22mmol) in dry MeCN (45 mL) was heated at reflux for1 h, then concentratedunder reduced pressure. The residue was partitioned between EtOAc andwater, and the organic layer was washed with water and evaporated. Theresidue was chromatographed on silic gel, eluting with CH₂Cl₂/EtOAc(1:4), followed by recrystallisation from MeOH/EtOAc/i-Pr₂O to giveIIa-14 (2.9 g, 81%): mp 142-143° C.; ¹H NMR [(CD₃)₂SO] δ 8.73 (t, J=5.7Hz, 1 H), 8.53 (s, 1 H), 7.38 (s, 1 H), 4.76 (t, J=5.5 Hz, 1 H), 3.68(t, J=6.9 Hz, 4 H), 3.57-3.49 (m, 2 H), 3.39 (t, J=6.9 Hz, 4 H),3.34-3.26 (m, partially obscured, 2 H). Anal. (C₁₃H₁₆I₂N₄O₆) C, H, N.

Example 9 2-(Aziridin-1-yl)-N-(6-Hydroxyhexyl)-3,5-Dinitrobenzamide(IIa-1)

A solution of 2-chloro-N-(6-hydroxyhexyl)-3,5-dinitrobenzamide (16) [forpreparation see Example 14 below] (118 mg, 0.34 mmol) and Et₃N (200 mg)in EtOAc (200 mL) was treated with aziridine (100 mg) at roomtemperature for 3 h. The mixture was diluted with EtOAc and washed threetimes with water, after dry, concentrated under reduced pressure untilabout 20 mL, the yellow solid was collected and gave 101 mg product(84%); ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.8 Hz, 1 H), 8.63 (m, 1 H), 8.29(d, J=2.8 Hz, 1 H), 4.31 (m, 1 H), 3.39 (m, 2 H), 3.25 (m, 2 H), 2.37(s, 4 H), 1.56 (m, 2 H), 1.43 (m, 2 H), 1.33 (m, 4 H). Anal.(C₁₅H₂₀N₄O₆) C, H, N.

Example 10 (Scheme 2d)2-[Bis(2-Chloroethyl)Amino]-N-(2-Hydroxyethyl)-3,5-Dinitrobenzamide(IIb-2) and2-[Bis(2-Bromoethyl)Amino]-N-(2-Hydroxyethyl)-3,5-Dinitrobenzamide(IIb-7)

2-Chloro-3,5-dinitrobenzoic acid (1) (18 g, 81 mmol) was treated withSOCl₂ (250 ml) containing one drop of DMF and heated under reflux for 6h. Evaporation of reagent followed by azeotroping with benzene gave thecrude acid chloride, which was dissolved in THF (200 mL) and addeddropwise to a solution made of 25 mL of 2-aminoethanol in THF (400 mL)and cooled with dryice-acetone bath. After stirring for 20 min. thereaction mixture was acidified to pH 4-5 with 1 N HCl, most of thesolvent was evaporated, and the residue was partitioned between water(250 mL) and EtOAc (300 mL). The aqueous phase was extracted with EtOAc,and the combined organic phases were washed with sat. NaHCO₃, 1 N HCland brine respectively, then concentrated to give2-chloro-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (2) 21.34 g (91%) as awhite solid: mp (EtOAc) 159-160° C.; ¹H NMR [(CD₃)₂SO] δ 8.99 (d, J=2.6Hz, 1 H, H-4), 8.86 (m, 1 H, CONH), 8.56 (d, J=2.6 Hz, 1 H, H-6), 4.83(m, 1 H, OH), 3.54 (m, 4 H). Anal. (C₉H₈ClN₃O₆) C, H, N.

A solution of 12 (1.52 g, 5.3 mmol) and Et₃N (4 mL) in p-dioxane (60 mL)was treated with N,N-bis(2-chloroethyl)amine hydrochloride (3.0 g, 16.5mmol) at 50° C. for 24 h. The mixture was poured into water andextracted with EtOAc to give the crude product, which waschromatographed on silica gel. Elution with EtOAc/petroleum ether (4:1)and concentration of the eluate under reduced pressure gave a oilyresidue that was dissolved in minimum amount of EtOAc. Petroleum etherwas added slowly until incipient cloudiness, and the solution was stoodovernight to precipitate2-[bis(2-chloroethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide(IIb-2) (2.07 g, 100%) as yellow crystals: mp (EtOAc/petroleum ether)109-111° C.; ¹H NMR [(CD₃)₂SO] δ8.73 (d, J=2.6 Hz, 1 H, H-4), 8.72 (m, 1H, CONH), 8.34 (d, J=2.6 Hz 1 H, H-6), 4.83 (m, 1 H, OH), 3.72 (m, 4 H,2xCH₂Cl), 3.55 (m, 2 H), 3.42 (m, 4 H, 2xCH₂N), 3.34 (m, 2 H); ¹³C NMR δ165.3, 145.8, 145.3, 141.0, 136.3, 127.5, 122.1, 59.1, 54.1, 42.1, 41.5.HRMS (FAB) [MH⁺] Calcd. For C₁₃H₁₇ ³⁵Cl₂N₄O₆ m/z 395.0525. Found;395.0525.

A solution of IIb-2 (1.20 g, 3.0 mmol) and LiBr (5.0 g, 58 mmol) in3-methyl-2-butanone (20 mL) was heated under reflux for 6 h, then cooledand poured into water. Extraction with EtOAc gave a crude product (<95%pure), that was re-treated with LiBr (5.0 g, 58 mmol) in3-methyl-2-butanone for a further 4 h, then worked up andchromatographed on silica gel, eluting with EtOAc/petroleum ether (from1:1 to 1:0), to give IIb-7 (1.39 g, 95%): mp (EtOAc/petroleum ether)105-108° C.; ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.7 Hz, 1 H, H-4), 8.73 (m,1 H, CONH), 8.34 (d, J=2.7 Hz, 1 H, H-6), 4.83 (m, 1 H, OH), 3.59-3.29(m, 12 H); ¹³C NMR δ 165.3, 145.4, 145.3, 141.1, 136.5, 127.4, 122.1,59.3, 53.9, 42.1, 30.0. HRMS (FAB) Calcd. For C₁₃H₁₇ ⁷⁹Br₂N₄O₆ [M+H^(+])m/z 482.9515. Found; 482.9492. Anal. (C₁₃H₁₆Br₂N₄O₆) H, N, Br; C: found,32.9; calculated 32.3%.

Example 11 (Scheme 2d)2-[Bis(2-Chloroethyl)Amino]-N-(3-Hydroxypropyl)-3,5-Dinitrobenzamide(IIb-3) and2-[Bis(2-Bromoethyl)Amino]-N-(3-Hydroxypropyl)-3,5-Dinitrobenzamide(IIb-8)

Reaction of the acid chloride of 11 (17 g) with 3-aminopropanol (7.5 g)in Me₂CO (120 mL) at 0° C. as described above, gave2-chloro-N-(3-hydroxypropyl)-3,5-dinitrobenzamide (13) (5.06 g, 26%): mp(EtOAc/petroleum ether) 120-121° C.; ¹H NMR [(CD₃)₂SO] δ 8.99 (d, J=2.6Hz, 1 H, H-4), 8.79 (m, 1 H, CONH), 8.51 (d, J=2.6 Hz, 1 H, H-6), 4.50(m, 1 H, OH), 3.49 (m, 2 H), 3.32 (m, 2 H), 1.70 (m, 2 H). Anal.(C₁₀H₁₀ClN₃O₆) C, H, N.

A solution of 13 (1.39 g, 4.58 mmol) and Et₃N (4 mL) in p-dioxane (60mL) was treated with N,N-bis(2-chloroethyl)amine hydrochloride (2.9 g,16.0 mmol) at 50 C. for 24 h. Workup as described above gave IIb-3 (1.84g, 100%): mp (EtOAc/petroleum ether) 89-91° C.; ¹H NMR [(CD₃)₂SO] δ8.74(d, J=2.7 Hz, 1 H, H-4), 8.71 (m, 1 H, CONH), 8.30 (d, J=2.7 Hz, 1 H,H-6), 4.52 (m, 1 H, OH), 3.71 (m, 4 H, 2xCH₂Cl), 3.50 (m, 2 H), 3.42 (m,4 H, 2xCH₂N), 3.32 (m, 2 H), 1.71 (m, 2 H); ¹³C NMR δ 165.1, 145.7,145.5, 141.0, 136.4, 127.3, 122.1, 58.4, 54.1, 41.5, 36.7, 31.8. HRMS(FAB) Calcd. For C₁₄H₁₉ ³⁵Cl₂N₄O₆ [M+H⁺]m/z 409.0682. Found; 409.0678.

Treatment of IIb-3 with LiBr in 3-methyl-2-butanone twice, as describedabove, gave IIb-8 (74% yield): mp (EtOAc/petroleum ether) 89-94° C.; ¹HNMR [(CD₃)₂SO] δ 8.74 (d, J=2.7 Hz, 1 H, H-4), 8.72 (m, 1 H, CONH), 8.30(d, J=2.7 Hz, 1 H, H-6), 3.77-3.44 (m, 12 H), 1.70 (m, 2 H); ¹³C NMR δ165.1, 145.5, 145.3, 141.2, 136.5, 127.3, 122.1, 58.4, 54.0, 36.7, 31.8,29.9. HRMS (FAB) Calcd. For C₁₄H₁₉ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 496.9671. Found;496.9658.

Example 12 (Scheme 2d)2-[Bis(2-Chloroethyl)Amino]-N-(4-Hydroxybutyl)-3,5-Dinitrobenzamide(IIb-4) and2-[Bis(2-Bromoethyl)Amino]-N-(4-Hydroxybutyl)-3,5-Dinitrobenzamide(IIb-9)

Reaction of the acid chloride of 11 (2.65 g, 10 mmol) with4-aminobutanol (1.9 g) as above, followed by acidification to pH 4-5with 1 N HCl and evaporation of most of the solvent gave a residue. Thiswas partitioned between water (50 mL) and EtOAc (100 mL). The aqueousphase was extracted with EtOAc, and the combined organic phase werewashed with sat. NaHCO₃, 1 N HCl and brine respectively, thenconcentrated to give 2-chloro-N-(4-hydroxybutyl)-3,5-dinitrobenzamide(14) 1.11 g (35%): mp (EtOAc) 121-124° C.; ¹H NMR [(CD₃)₂SO] δ 8.98 (d,J =2.7 Hz, 1 H), 8.79 (m, 1 H), 8.52 (d, J=2.7 Hz, 1 H), 4.43 (m, 1 H),3.43 (m, 2 H), 3.26 (m, 2 H), 1.54 (m, 4 H); ¹³C NMR δ 162.6, 148.4,145.9, 140.4, 128.2, 125.8, 120.4, 60.2, 39.1, 29.8, 25.3. Anal.(C₁₁H₁₂ClN₃O₆) C, H, N.

A solution of 14 (0.75 g, 2.3 mmol) and Et₃N (2 mL) in p-dioxane (30 mL)was treated with N,N-bis(2-chloroethyl)amine hydrochloride (1.5 g, 8.0mmol) at 50° C. for 24 h. The mixture was poured into water andextracted with EtOAc gave the crude product which was chromatographed onsilica gel. Elution with EtOAc/petroleum ether (4:1) gave IIb-4 (0.99 g,100%) as yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.71 (d, J=2.8 Hz, 1 H), 8.69(m, 1 H), 8.27 (d, J=2.8 Hz, 1 H), 4.37 (m, 1 H), 3.70 (m, 4 H), 3.38(m, 6 H), 3.25 (m, 2 H), 1.56 (m, 2 H), 1.47 (m, 2 H); ¹³C NMR δ 165.0,145.7, 145.5, 141.0, 136.4, 127.2, 122.0, 60.2, 54.2, 41.5, 39.2, 29.8,25.2. HRMS (FAB) Calcd. For C₁₅H₂₁ ³⁵Cl₂N₄O₆ [M+H⁺] m/z 423.0838. Found;423.0847.

A solution of IIb-4 (0.96 g, 3.04 mmol) and LiBr (5 g) in3-methyl-2-butanone (15 mL) was heated under reflux for 6 h, then cooledand poured into water. Extraction with EtOAc gave a crude product (<95%pure), that was re-treated with LiBr (5 g) in 3-methyl-2-butanone for afurther 4 h, then worked up and chromatographed on silica gel, elutingwith EtOAc/petroleum ether (from 1:1 to 3:1) give IIb-9 (1.01 g, 87%) asa yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.8 Hz, 1 H), 8.72 (m, 1H), 8.28 (d, J=2.8 Hz, 1 H), 3.60-3.26 (m, 12 H), 1.58 (m, 2 H), 1.49(m, 2 H); ¹³C NMR δ 165.0, 145.6, 145.2, 141.2, 136.5, 127.2, 122.0,60.2, 54.1, 39.2, 29.9, 29.8, 25.2. HRMS (FAB) Calcd. For C₁₅H₂₁⁷⁹Br₂N₄O₆ [M+H⁺] m/z 510.9828. Found; 510.9832.

Example 13 (Scheme 2d)2-[Bis(2-Chloroethyl)Amino]-N-(5-Hydroxypentyl)-3,5-Dinitrobenzamide(IIb-5) and2-[Bis(2-Bromoethyl)Amino]-N-(5-Hydroxypentyl)-3,5-Dinitrobenzamide(IIb-10)

Similar reaction of the acid chloride of 11 with 5-aminopentanol asabove gave 2-chloro-N-(5-hydroxypentyl)-3,5-dinitrobenzamide (15), 1.3 g(39%), mp (EtOAc) 105-108° C.; ¹H NMR [(CD₃)₂SO] δ 8.98 (d, J=2.7 Hz, 1H), 8.79 (m, 1 H), 8.50 (d, J=2.7 Hz, 1 H), 4.35 (m, 1 H), 3.39 (m, 2H), 3.26 (m, 2 H), 1.54 (m, 2 H), 1.44 (m, 2 H), 1.36 (m, 2 H); ¹³C NMRδ 162.7, 148.4, 145.9, 140.4, 128.2, 125.8, 120.4, 60.5, 39.1, 32.0,28.4, 22.8. Anal. (C₁₂H₁₄ClN₃O₆) C, H, N.

A solution of 15 (0.63 g, 2.3 mmol) and Et₃N (2 mL) in p-dioxane (30 mL)was treated with N,N-bis(2-chloroethyl)amine hydrochloride (1.5 g, 8.0mmol) at 50° C. for 24 h. The mixture was poured into water andextracted with EtOAc to gave the crude product which was chromatographedon silica gel. Elution with EtOAc/petroleum ether (4:1) gave IIb-5 (0.82g, 100%) as yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.73 (d, J=2.8 Hz, 1 H),8.69 (m, 1 H), 8.28 (d, J=2.8 Hz, 1 H), 4.32 (m, 1 H), 3.70 (m, 4 H),3.40 (m, 6 H), 3.25 (m, 2 H), 1.55 (m, 2 H), 1.47 (m, 2 H), 1.37 (m, 2H); ¹³C NMR δ 165.0, 145.7, 145.5, 141.0, 136.4, 127.2, 122.0, 60.5,54.2, 41.5, 39.3, 32.0, 28.3, 22.9. HRMS (FAB) Calcd. For C₁₆H₂₃³⁵Cl₂N₄O₆ [M+H⁺] m/z 437.0995. Found; 437.0991.

Similar reaction of IIb-5 (1.3 g) with LiBr gave IIb-10 (1.35 g, 86%) asa yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.8 Hz, 1 H), 8.71 (m, 1H), 8.28 (d, J=2.8 Hz, 1 H), 3.60-3.26 (m, 12 H), 1.55 (m, 2 H), 1.48(m, 2 H), 1.37 (m, 2 H); ¹³C NMR δ 165.0, 145.6, 145.2, 141.2, 136.5,127.2, 122.0, 60.5, 54.1, 39.3, 32.0, 29.8, 28.4, 22.9. HRMS (FAB)Calcd. For C₁₆H₂₃ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 524.9984. Found; 524.9975.

Example 14 (Scheme 2d)2-[Bis(2-Chloroethyl)Amino]-N-(6-Hydroxybexyl)-3,5-Dinitrobenzamide(IIb-6) and2-[Bis(2-Bromoethyl)Amino]-N-(6-Hydroxyhexyl)-3,5-Dinitrobenzamide(IIb-11)

Similar reaction of the acid chloride of 11 with 6-aminohexanol as abovegave 2-chloro-N-(6-hydroxyhexyl)-3,5-dinitrobenzamide (16), 0.9 g (26%),mp (EtOAc) 88-91° C.; ¹H NMR [(CD₃)₂SO] δ 8.98 (d, J=2.7 Hz, 1 H), 8.78(m, 1 H), 8.49 (d, J=2.7 Hz, 1 H), 4.32 (m, 1 ), 3.39 (m, 2 H), 3.26 (m,2 H), 1.54 (m, 2 H), 1.44 (m, 2 H), 1.34 (m, 4 H); ¹³C NMR δ 162.7,148.4, 145.9, 140.4, 128.2, 125.8, 120.4, 60.5, 39.1, 32.3, 28.6, 26.2,25.1. Anal. (C₁₃H₁₆ClN₃O₆) C, H, N.

A solution of 16 (0.67 g, 2.5 mmol) and Et₃N (2 mL) in p-dioxane (30 mL)was treated with N,N-bis(2-chloroethyl)amine hydrochloride (1.5 g, 8.0mmol) at 50° C. for 24 h. The mixture was poured into water andextracted with EtOAc to gave the crude product which was chromatographedon silica gel. Elution with EtOAc/petroleum ether (4:1) gave IIb-6 (0.87g, 100%) as yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.73 (d, J=2.8 Hz, 1 H),8.70 (m, 1 H), 8.28 (d, J=2.8 Hz, 1 H), 4.31 (m, 1 H), 3.70 (m, 4 H),3.38 (m, 6 H), 3.25 (m, 2 H), 1.54 (m, 2 H), 1.40 (m, 2 H), 1.32 (m, 4H); ¹³C NMR δ 165.0, 145.7, 145.6, 141.0, 136.4, 127.2, 122.0, 60.5,54.2, 41.5, 39.2, 32.3, 28.5, 26.3, 25.1. HRMS (FAB) Calcd. For C₁₇H₂₅³⁵Cl₂N₄O₆ [M+H⁺] m/z 451.1151. Found; 451.1154.

Similar reaction of IIb-6 (0.97 g) with LiBr gave IIb-11 (0.96 g, 81%)as a yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.8 Hz, 1 H), 8.70 (m,1 H), 8.28 (d, J=2.8 Hz, 1 H), 3.60-3.26 (m, 12 H), 1.54 (m, 2 H), 1.43(m, 2 H), 1.32 (m, 4 H); ¹³C NMR δ 165.0, 145.6, 145.2, 141.2, 136.5,127.2, 122.0, 60.6, 54.1, 39.2, 32.4, 29.9, 28.5, 26.3, 25.1. HRMS (FAB)Calcd. For C₁₇H₂₅ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 539.0141. Found; 539.0135.

Example 15 (Scheme 2e)2-[Bis(2-Bromopropyl)Amino]-N-(2-Hydroxyethyl)-3,5-Dinitrobenzamide(IIb-7a)

Reaction of2-chloro-3,5-dinitro-N-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]benzamide(17) (1.02 g) [For method of preparation see co-pending NZ ApplicationNo. 521851] with diisopropanolamine (0.8 g) as above gave2-[bis(2-hydroxypropanyl)amino]-3,5-dinitro-N-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]benzamide(18) (1.29 g, 100%): as a yellow foam; ¹H NMR [(CD₃)₂SO] δ 9.22 (br, 1H), 8.66 (d, J=2.8 Hz, 1 H), 8.29 (d, J=2.8 Hz, 1 H), 4.99 (m, 1 H),4.85 (br, 1 H), 4.62 (br, 1 H), 3.94 (m, 2 H), 3.77 (m, 2 H), 3.53 (m, 4H), 3.26 (m, 2 H), 1.48 (m, 10 H), 0.98 (m, 6 H); ¹³C NMR δ 166.5,147.8, 142.4, 138.2, 132.6, 128.8, 123.8, 98.1, 64.8, 63.5, 61.5, 60.1,30.1, 25.0, 20.5, 20.2, 19.1. HRMS (FAB) Calcd. For C₂₀H₃₁N₄O₉ [M+H⁺]m/z 471.2091. Found; 471.2089.

Reaction of 18 with MsCl as above gave1-methyl-2-[{2-[(methylsulfonyl)oxy]propyl}-2,4-dinitro-6-({[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]amino}carbonyl)anilino]ethylmethanesulfonate (19) (2.52 g, 100%): as a yellow foam; which was useddirectly for the next step.

A solution of 19 (2.52 g, 4.03 mmol) in THF (150 mL) was treated with1 NHCl (100 mL), and the solution was stirred at 20° C. for 1 h, thendiluted with water (100 mL), neutralized with satd. NaHCO₃, andextracted with EtOAc (3×80 mL). The combined organic phases were washedwith brine and dried, the solvent was evaporated, and the residue waspurified by chromatography on silica gel, eluting with EtOAc/MeOH(100:1), to give2-(2-{[(2-hydroxyethyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]propyl}-4,6-dinitroanilino)-1-methylethylmethanesulfonate (20) (0.80 g, 37%): as a yellow foam; ¹H NMR [(CD₃)₂SO]δ 8.94 (m, 1 H), 8.72 (m, 1 H), 8.35 (m, 1 H), 4.92 (m, 2 H), 3.56 (m, 2H), 3.30 (m, 6 H), 3.16 (s, 6 H), 1.32 (m, 6 H); ¹³C NMR δ 165.9, 145.8,143.4, 139.4, 133.6, 128.0, 123.1, 76.3, 59.2, 57.3, 42.2, 37.7, 18.6.HRMS (FAB) Calcd. For C₁₇H₂₇N₄O₁₂S₂ [M+H⁺] m/z 543.1067. Found;543.1074.

Treatment of 20 (0.52 g, 0.96 mmol) with LiBr (0.5 g, 5.8 mmol) in EtOAc(50 mL) at 60° C. for 3 h, and chromatography of the product on silicagel, eluting with EtOAc/petroleum ether (from 2:1 to 1:0) gave IIb-7a(0.31 g, 62%): as yellow solid: mp (EtOAc/petroleum ether) 127-130° C.;¹H NMR [(CD₃)₂SO] δ 8.91 (m, 1 H, CONH), 8.70 (d, J=2.8 Hz, 1 H, H-4),8.32 (d, J=2.8 Hz, 1 H, H-6), 4.80 (m, 1 H), 4.42 (m, 2 H), 3.55 (m, 4H), 1.62 (m, 6 H); ¹³C NMR δ 165.8, 144.8, 143.5, 139.6, 133.6, 128.0,122.9, 60.6, 59.2, 47.9, 42.2, 23.4. Anal. (C₁₅H₂₀Br₂N₄O₆) C, H, N.

Example 16 (Scheme 2f)2-((2-Bromoethyl)-2-{[(2-Hydroxypropyl)Amino]Carbonyl}-4,6-Dinitroanilino)EthylMethanesulfonate (IIb-13)

A solution of 13 (1.22 g, 4.0 mmol) in 50 mL of CH₂Cl₂ was cooled in anice-bath, and 3,4-dihydro-2H-pyran (1.0 mL) and p-toluenesulfonic acid(0.1 g) were added. The reaction mixture was stirred for 2 h, thenconcentrated under reduced pressure. Chromatography of the residue onsilica gel, eluting with EtOAc/petroleum ether (from 1:2 to 2:1), gave2-chloro-3,5-dinitro-N-[2-(tetrahydro-2H-pyran-2-yloxy)propyl]benzamide(21) (1.45 g, 94%): as a pale yellow oil; ¹H NMR [(CD₃)₂SO] δ 8.99 (d,J=2.7 Hz, 1 H, H-4), 8.81 (m, 1 H, CONH), 8.51 (d, J=2.7 Hz, 1 H, H-6),4.57 (m, 1 H), 3.72 (m, 2 H), 3.46-3.25 (m, 4 H), 1.82-1.44 (m, 8 H).¹³C NMR δ 162.7, 148.4, 145.9, 140.3, 128.2, 125.8, 120.5, 98.0, 64.2,61.3, 36.5, 30.2, 28.9, 24.9, 19.1. HRMS (FAB) Calcd. For C₁₅H₁₉³⁵ClIN₃O₇ [M+H⁺] m/z 388.0912. Found; 388.0915.

Reaction of 21 (1.45 g, 3.75 mmol) with diethanolamine (1.67 g) as abovegave2-[bis(2-hydroxyethyl)amino]-3,5-dinitro-N-[2-(tetrahydro-2H-pyran-2-yloxy)propyl]benzamide(22) (1.62 g, 95%): as a yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.96 (m, 1 H,CONH), 8.66 (d, J=2.8 Hz, 1 H, H-4), 8.31 (d, J=2.8 Hz, 1 H, H-6), 4.95(m, 2 H), 4.56 (m, 1 H), 3.79-3.16 (m, 14 H), 1.80-1.45 (m, 8 H); ¹³CNMR δ 166.2, 148.1, 143.6, 139.3, 133.8, 128.9, 123.8, 98.5, 64.8, 61.7,58.5, 54.6, 37.3, 30.6,29.2, 25.4, 19.6. HRMS (FAB) Calcd. ForC₁₉H₂₉N₄O₆ [M+H⁺] m/z 457.1935. Found; 457.1939.

Reaction of 22 (1.62 g, 3.55 mmol) with MsCl (2 mL) as above gave2-[{2-[(methylsulfonyl)oxy]ethyl}-5,6-dinitro-6-({[2-(tetrahydro-2H-pyran-2-yloxy)propyl]-amino}carbonyl)anilino]ethylmethanesulfonate (23) (2.17 g, 100%): as a yellow foam; ¹H NMR[(CD₃)₂SO] δ 8.71 (d, J=2.8 Hz, 1 H), 8.71 (m, 1 H), 8.31 (d, J=2.8 Hz,1 H), 4.26 (m, 4 H), 3.71-3.37 (m, 10 H), 3.13 (s, 6 H), 3.10 (m, 2 H),1.82-1.43 (m, 8 H); ¹³C NMR δ 165.1, 146.3, 145.4, 140.9, 135.9, 127.4,122.2, 98.0, 67.2, 64.3, 51.4, 45.7, 36.5, 30.2, 28.7, 24.9, 19.1, 8.5.HRMS (FAB) Calcd. For C₂₁H₃₃N₄O₁₃S₂ [M+H⁺] m/z 613.1486. Found;613.1481.

A solution of 23 (2.95 g, 3.55 mmol) in THF (120 mL) was treated with 1N HCl (80 mL), and the solution was stirred at 20° C. for 1 h, thendiluted with water (100 mL), neutralized with satd. NaHCO₃, andextracted with EtOAc (3×80 mL). The combined organic phases were washedwith brine and dried, the solvent was evaporated, and the residue waspurified by chromatography on silica gel, eluting with EtOAc/MeOH(100:1), to give2-(2-{[(3-hydroxypropyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-4,6-dinitroanilino)ethylmethanesulfonate (24) (1.4 g, 75%): as a yellow solid: mp(EtOAc/petroleum ether) 130-133° C.; ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.8Hz, 1 H), 8.72 (m, 1 H), 8.32 (d, J=2.8 Hz, 1 H), 4.29 (m, 4 H), 3.47(m, 8 H), 3.14 (s, 6 H), 1.71 (m, 2 H); ¹³C NMR δ 165.2, 146.3, 145.3,140.8, 135.9, 127.5, 122.3, 67.3, 58.4, 51.4, 36.8, 36.5, 31.7. Anal.(C₁₆H₂₄N₄O₁₂S₂) C, H, N.

Treatment of 24 (0.25 g, 0.45 mmol) with LiBr (53 mg, 0.61 mmol) inEtOAc (50 mL) at 60° C. for 3 h, and chromatography of the product onsilica gel, eluting with EtOAc/petroleum ether (from 2:1 to 1:0) gaveIIb-13 (0.16 g, 66%): as yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.74 (d, J=2.8Hz, 1 H), 8.73 (m, 1 H), 8.31 (d, J=2.8 Hz, 1 H), 4.28 (m, 2 H),3.65-3.44 (m, 10 H), 3.13 (s, 3 H), 1.70 (m, 2 H); ¹³C NMR δ 165.1,145.7, 145.4, 141.0, 136.2, 127.3, 122.1, 67.5, 58.4, 51.1, 36.7, 36.5,31.7, 29.6. HRMS (FAB) Calcd. For C₁₅H₂₂ ⁷⁹BrN₄O₉S [M+H⁺] m/z 513.0291.Found; 513.0281.

Example 17 (Scheme 2g)2-((2-Bromoethyl)-2-{[(2-Hydroxyethyl)Amino]Carbonyl}-4,6-Dinitroanilino)EthylMethanesulfonate (IIb-12)

Solid IIb-7 (300 mg, 0.62 mmol) and silver methanesulfonate (130 mg,0.65 mmol) in dry MeCN (15 mL) were heated under reflux for 3 h, thencooled and filtered. The solid AgBr was washed with EtOAc to give a 98%yield of AgBr. The solvent was removed at reduced pressure and theresidue was separated by chromatography on silica gel, eluting withEtOAc/petroleum ether (from 1:1 to 1:0), to give successively:

-   -   starting material (IIb-7) (28 mg, 9%).    -   IIb-12 (123 mg, 38%) as a yellow foam; ¹H NMR [(CD₃)₂SO] δ 8.77        (m, 1 H, CONH), 8.74 (d, J=2.7 Hz, 1 H, H-4), 8.36 (d, J=2.7 Hz,        1 H, H-6), 4.28 (m, 2 H, CH₂OMs), 3.58 (m, 4 H), 3.44 (m, 4 H),        3.14 (s, 3 H, OSO₂CH₃); ¹³C NMR δ 165.3, 145.8, 145.2, 140,9,        135.1, 127.5, 122.2, 67.5, 59.2, 54.2, 51.0, 42.1, 36.4, 29.7;        HRMS m/z required for C₁₄H₂₀ ⁷⁹BrN₄O₉S 499.01344; Found        499.01324.

The column was finally eluted with EtOAc/MeOH (9:1) to give2-(2-{[(2-hydroxyethyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-4,6-dinitroanilino)ethylmethanesulfonate 25 (159 mg, 53%) as a yellow solid: mp 128-132° C.(EtOAc/petroleum ether); ¹H NMR [(CD₃)₂SO] δ 8.78 (m, 1 H, CONH), 8.74(d, J=2.7 Hz, 1 H, H-4), 8.36 (d, J=2.7 Hz, 1 H, H-6), 4.29 (m, 4 H,2xCH₂OMs), 3.56 (m, 2 H), 3.45 (m, 6 H), 3.14 (s, 6 H, 2xOSO₂CH₃); ¹³CNMR δ 165.4, 146.3, 145.1, 140,6, 135.8, 127.6, 122.3, 67.3, 59.2, 51.3,42.1,36.4; HRMS: C₁₅H₂₃N₄O₁₂S₂ requires m/z 515.0754. Found: 515.0744.

Example 18 (Scheme 2h)2-((2-Chloroethyl)-2-{[(2-Hydroxyethyl)Amino]Carbonyl}-4,6-Dinitroanilino)EthylMethanesulfonate (IIb-2m)

A solution of 25 (5.3 g, 10.3 mmol) in DMF (100 mL) was treated withLiCl (524 mg, 12.4 mmol) at 60° C. for 2 h, and then cooled, poured intodilute HCl and extracted with EtOAc (3×150 mL). Workup andchromatography of the product on silica gel, eluting withEtOAc/petroleum ether from 1:1 to 1:0, gave IIb-2 (2.4 g, 59%), and thenIIb-2m (1.94 g, 41%) as yellow oil; ¹H NMR [(CD₃)₂SO] δ 8.77 (m, 1 H,CONH), 8.74 (d, J=2.7 Hz, 1 H, H-4), 8.36 (d, J=2.7 Hz, 1 H, H-6), 4.28(m, 2 H, —CH₂O-Ms), 3.58 (m, 4 H), 3.44 (m, 4 H), 3.14 (s, 3 H,—OSO₂CH₃); ¹³C NMR δ 165.3, 145.8, 145.2, 140,9, 135.1, 127.5, 122.2,67.5, 59.2, 54.2, 51.0, 42.1, 36.4, 29.7.

IIb-2m was prepared by an alternative method as following: A solution ofIIb-2 (12.50 g, 31.6 mmol) in 3-methyl-2-butanone (150 mL) was treatedat 25° C. with NaI (5.69 g, 38.0 mmol) and the mixture was stirred at70° C. for 2 h and then concentrated under reduced pressure. The residuewas partitioned between water (250 mL) and EtOAc (250 mL) and theseparated organic layer was washed with water, dried (Na₂SO₄) and thenconcentrated under reduced pressure. The resulting oil (15.23 g) wasdissolved in CH₃CN (80 mL), treated with silver methanesulfonate (9.63g, 47.4 mmol) and the mixture was stirred at 25° C. for 1 h and thenconcentrated under reduced pressure. The residue was extracted withEtOAc (200 mL), filtered, the solids were washed with EtOAc (100 mL) andthe EtOAc solution was evaporated and the oily mixture was separated bychromatography on silica gel as above and gave starting material (3.61g, 29%), IIb-2m (4.55 g, 32%) and 25 (4.98 g, 31%). When NaI wasreplaced by LiBr the reaction gave a similar result.

Example 19 (Scheme 2i)2-[Bis(2-Iodoethyl)Amino]-N-(2-Hydroxyethyl)-3,5-Dinitrobenzamide(IIb-14) and2-((2-Iodoethyl)-2-{[(2-Hydroxyethyl)Amino]Carbonyl}-4,6-Dinitroanilino)EthylMethanesulfonate (IIb-15)

Treatment of 25 (6.7 g, 13.0 mmol) with NaI (2.9 g, 20 mmol) in EtOAc(200 mL) at 60° C. for 3 h, and chromatography of the product on silicagel, eluting with EtOAc/petroleum ether (from 2:1 to 1:0) gave IIb-14(3.3 g, 44%) as a yellow solid: mp (EtOAc/petroleum ether) 129-131° C.;¹H NMR [(CD₃)₂SO] δ 8.72 (d, J=2.8 Hz, 1 H, H-4), 8.70 (m, 1 H, CONH),8.32 (d, J=2.8 Hz, 1 H, H-6), 4.80 (m, 1 H), 3.55 (m, 2 H), 3.43 (m, 4H), 3.31 (m, 6 H); ¹³C NMR δ 165.3, 145.2, 144.7, 141.0, 136.3, 127.3,122.0, 59.3, 54.7, 42.1, 2.94. Anal (C₁₃H₁₆N₄I₂O₆) C, H, N.

Later eluates gave IIb-15 (1.35 g, 19%) as a yellow foam; ¹H NMR[(CD₃)₂SO] δ 8.74 (d, J=2.8 Hz, 1 H, H-4), 8.74 (m, 1 H, CONH), 8.34 (d,J=2.8 Hz, 1 H, H-6), 4.28 (m, 2 H), 3.56 (m, 2 H), 3.43 (m, 2 H), 3.31(m, 6 H), 3.13 (s, 3 H); ¹³C NMR δ 165.3, 145.5, 145.2, 140.8, 136.1,127.4, 122.1, 67.5, 59.2, 55.4, 50.6, 42.1, 36.5, 2.6. HRMS (FAB) Calcd.For C₁₄H₂₀IN₄O₉S [M+H⁺] m/z 546.9996. Found; 546.9997.

Example 20 (Scheme 2j)3-[Bis(2-Bromoethyl)Amino]-N-(2-Hydroxyethyl)-2,6-Dinitrobenzamide(IIc-7) and2-((2-Bromoethyl)-3-{[(2-Hydroxyethyl)Amino]carbonyl}-2,4-Dinitroanilino)EthylMethanesulfonate (IIc-12)

Treatment of2-(3-{[(2-hydroxyethyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethylmethanesulfonate (26) [for method of preparation see NZ Application No.521851] (310 mg, 0.6 mmol) in EtOAc (50 mL) with LiBr (78 mg, 0.9 mmol),followed by chromatography on silica gel and elution withEtOAc/petroleum ether (from 1:1 to 1:0) gave IIc-7 (70 mg, 25%) as afoam; ¹H NMR [(CD₃)₂SO] δ 8.80 (m, 1 H, CONH), 8.24 (d, J=9.4 Hz, 1 H),7.63 (d, J=9.4 Hz, 1 H), 4.66 (m, 1 H), 3.70 (m, 4 H), 3.60 (m, 4 H),3.45 (m, 2 H), 3.22 (m, 2 H); ¹³C NMR δ 161.4, 145.8, 140.2, 137.5,129.2, 127.6, 122.6, 59.0, 52.6, 41.7, 30.0. HRMS (FAB) Calcd. ForC₁₃H₁₇ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 482.9515. Found; 482.9508.

Further elution with EtOAc/MeOH (50:2) gave IIc-12 (118 mg, 39%): mp.94-97° C,; ¹H NMR [(CD₃)₂SO] δ 8.80 (m, 1 H, CONH), 8.25 (d, J=9.4 Hz, 1H), 7.64 (d, J=9.4 Hz, 1 H), 4.67 (m, 1 H), 4.27 (m, 2 H), 3.63 (m, 4H), 3.57 (m, 2 H), 3.45 (m, 2 H), 3.26 (m, 2 H), 3.15 (s, 3 H); ¹³C NMRδ 161.4, 146.2, 140.5, 137.7, 129.2, 127.5, 122.9, 66.8, 59.0, 50.0,41.7,36.6, 29.9. Anal. (C₁₄H₁₉BrN₄O₉S) C, H, N.

Example 21 (Scheme 2j)3-[Bis(2-Bromoethyl)Amino]-N-(3-Hydroxypropyl)-2,6-Dinitrobenzamide(IIc-8) and2-((2-Bromoethyl)-3-{[(3-Hydroxypropyl)Amino]Carbonyl}-2,4-Dinitroanilino)EthylMethanesulfonate (IIc-13)

Treatment of2-(3-{[(3-hydroxypropyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethylmethanesulfonatee (27) [for method of preparation see co-pending NZApplication No. 521851] (716 mg, 1.36 mmol) in EtOAc (200 mL) with LiBr(175 mg, 2.0 mmol), followed by chromatography on silica gel and elutionwith EtOAc/petroleum ether (from 1:1 to 1:0) gave IIc-8 (289 mg, 42%) asa yellow solid; mp (EtOAc/petroleum ether) 142-144° C.; ¹H NMR[(CD₃)₂SO] δ 8.75 (t, J=5.8 Hz, 1 H, CONH), 8.23 (d, J=9.4 Hz, 1 H,H-5), 7.62 (d, J=9.4 Hz, 1 H, H-6), 4.47 (m, 1 H, CHOH), 3.68 (m, 4 H),3.57 (m, 4 H), 3.43 (m, 2 H), 3.20 (m, 2 H), 1.60 (m, 2 H); ¹³C NMR δ161.2, 146.9, 140.2, 137.5, 129.4, 127.7, 122.6, 58.3, 52.6, 36.4, 31.6,30.1. HRMS (FAB) Calcd. For C₁₄H₁₉ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 496.9671. Found:496.9667.

Further elution with EtOAc/MeOH (50:2) gave IIc-13 (270 mg, 39%): mp.115-117° C.; ¹H NMR [(CD₃)₂SO] δ 8.75 (t, J=5.8 Hz, 1 H, CONH), 8.24 (d,J=9.4 Hz, 1 H, H-5), 7.64 (d, J=9.4 Hz, 1 H, H-6), 4.43 (m, 1 H, CHOH),4.27 (m, 2 H, CH₂OMs), 3.66 (m, 4 H, 2xCH₂N), 3.59 (m, 2 H), 3.44 (m, 2H), 3.22 (m, 2 H), 3.15 (s, 3 H, CH₃SO₃), 1.60 (m, 2 H); ¹³C NMR δ161.1, 146.2, 140.5, 137.7, 129.2, 127.6, 122.9, 66.8, 58.2, 52.9, 50.0,36.6, 36.4, 31.6, 30.0. Anal. (C₁₅H₂₁BrN₄O₉S) C, H, N.

Example 22 (Scheme 2j)3-[Bis(2-Bromoethyl)Amino]-N-(4-Hydroxybutyl)-2,6-Dinitrobenzamide(IIc-9) and2-((2-Bromoethyl)-3-{[(4-Hydroxybutyl)Amino]Carbonyl}-2,4-Dinitroanilino)EthylMethanesulfonate (IIc-14)

Treatment of2-(3-{[(4-hydroxybutyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethylmethanesulfonate (28) [for method of preparation see NZ Application No.521851] (500 mg, 0.92 mmol) in EtOAc (100 mL) with LiBr (110 mg, 1.4mmol), followed by chromatography on silica gel and elution withEtOAc/petroleum ether (from 1:1 to 1:0) gave IIc-9 (100 mg, 21%) as afoam; ¹H NMR [(CD₃)₂SO] δ 8.73 (m, 1 H, CONH), 8.25 (d, J=9.4 Hz, 1 H),7.63 (d, J=9.4 Hz, 1 H), 4.38 (m, 1 H), 3.69 (m, 4 H), 3.57 (m, 4 H),3.40 (m, 2 H), 3.14 (m, 2 H), 1.47 (m, 4 H); ¹³C NMR δ 161.0, 145.8,140.2, 137.6, 129.3, 127.6, 122.6, 60.2, 52.6, 30.0, 29.6, 24.8. HRMS(FAB) Calcd. For C₁₅H₂₀ ⁷⁹Br₂N₄O₆ [M+H⁺] m/z 510.9828. Found; 510.9819.

Further elution with EtOAc/MeOH (50:2) gave IIc-14 (117 mg, 30%): mp.114-117° C.; ¹H NMR [(CD₃)₂SO] δ 8.74 (m, 1 H, CONH), 8.25 (d, J=9.4 Hz,1 H), 7.65 (d, J=9.4 Hz, 1 H), 4.37 (m, 1 H), 4.27 (m, 2 H), 3.65 (m, 4H), 3.57 (m, 2 H), 3.35 (m, 2 H), 3.16 (m,2 H), 3.15 (s, 3 H), 1.47 (m,4 H); ¹³C NMR δ 160.0, 146.1, 140.6, 137.8, 129.2, 127.5, 122.9, 66.8,60.2, 52.9, 50.0, 36.6, 29.9, 29.6, 24.9. Anal. (C₁₆H₂₃BrN₄O₉S) C, H, N.

Example 23 (Scheme 2k)2-(3-{[(3-Hydroxypropyl)Amino]Carbonyl{}2-[(Methylsulfonyl)Oxy]Ethyl}-2,4-Dinitroanilino)EthylMethanesulfonate (27) and2-((2-Bromoethyl)-3-{[(3-Hydroxypropyl)Amino]Carbonyl}-2,4-Dinitroanilino)EthylMethanesulfonate (IIc-13)

Solid IIc-8 (2.15 g, 4.3 mmol) was added to a hot solution of silvermethanesulfonate (0.992 g, 4.9 mmol) in dry MeCN (40 mL). The mixturewas heated under reflux for 3 h, then cooled and filtered. The solventwas removed at reduced pressure and the residue was separated bychromatography on silica gel, eluting with EtOAc/petroleum ether (from1:1 to 1:0), to give successively IIc-13 (0.5 g, 25%), IIc-8 (0.3 g,14%) and 27 (0.4 g, 18%).

Example 24 (Scheme 2k)2-((2-Chloroethyl)-3-{[(3-Hydroxypropyl)Amino]Carbonyl}-2,4-Dinitroanilino)EthylMethanesulfonate (IIc-6)

A solution of 27 (9.0 g, 17.0 mmol) in DMF (110 mL) was treated withLiCl (860 mg, 20.4 mmol) at 60° C. for 2 h, then cooled, poured intodilute HCl, and extracted with EtOAc (3×150 mL). Workup andchromatography of the product on silica gel, eluting withEtOAc/petroleum ether from 1:1 to 1:0, gave IIc-6 (4.0 g, 50%) as yellowcrystals: mp 104-109° C.; ¹H NMR [(CD₃)₂SO] δ 8.75 (t, J=5.8 Hz, 1 H,CONH), 8.24 (d, J =9.4 Hz, 1 H, H-5), 7.64 (d, J=9.4 Hz, 1 H, H-6), 4.44(m, 1 H, CHOH), 4.26 (m, 2 H), 3.72 (m, 2 H), 3.65 (m, 2 H), 3.59 (m, 2H), 3.43 (m, 2 H), 3.20 (m, 2 H), 3.15 (s, 3 H), 1.60 (m, 2 H); ¹³C NMRδ 161.1, 146.4, 140.5, 137.7, 129.2, 127.6, 122.9, 66.8, 58.2, 52.9,50.1, 41.4, 36.6, 36.4, 31.6. Anal. (C₁₅H₂₁ClN₄O₉S) C, H, N.

Example 25 (Scheme 2k)2-((2-Iodoethyl)-3-{[(3-Hydroxypropyl)Amino]Carbonyl}-2,4-Dinitroanilino)EthylMethanesulfonate (IIc-15)

A solution of 27 (5.28 g, 10.0 mmol) in EtOAc (250 mL) was treated withNaI (1.8 g, 12.0 mmol) at 60° C. for 2 h, and the product waschromatographed on silica gel, eluting with EtOAc/petroleum ether from1:2 to 1:0, to give IIc-15 (2.29 g, 41%) as yellow crystals: mp 100-103°C.; ¹H NMR [(CD₃)₂SO] δ 10.05 (s, 1 H), 7.40 (d, J=11.5 Hz, 1 H), 7.09(s, 1 H), 6.70 (d, J=11.5 Hz, 1 H), 2.50 (m, 2 H), 2.21 (m, 2 H), 2.03(s, 3 H), 1.52 (m, 4 H); ¹³C NMR δ 161.1, 145.8, 140.5, 137.7, 129.2,127.6, 122.9, 66.8, 58.2, 53.9, 49.9, 41.4, 36.6, 36.4, 31.6. Anal.(C₁₅H₂₁IN₄O₉S) C, H, N.

Preparation of Phosphates (Scheme 3)

Example 26 2-[[2-[Bis(2-Bromoethyl)Amino]-3,5-Dinitrobenzoyl]Amino]EthylDihydrogen Phosphate (Ib-7P)

A solution of alcohol IIb-7 (2.58 g, 5.33 mmol) and di-tert-butyldiethylphosphoramidite (93%, 2.0 mL, 6.9 mmol) in dry DMF (20 mL) underN₂ was treated with 1H-tetrazole (3 wt. % in CH₃CN, 55 mL, 18.7 mmol)and stirred at 20° C. for 1.5 h. The reaction mixture was then cooled to−50° C. and a solution of 3-chloroperoxybenzoic acid (55%, 2.68 g, 8.54mmol) was rapidly added such that the temperature was kept below −5° C.The reaction mixture was warmed to room temperature and diluted withCH₂Cl₂ (150 mL). The solution was washed with 5% aqueous Na₂S₂O₅ (2×50mL), 10% aqueous NaHCO₃ (2×50 mL), water (2×50 mL), dried, concentratedunder reduced pressure below 30° C. and the residue was shaken withi-Pr₂O/hexane and refrigerated. The resulting solid was purified bychromatography on silica gel, eluting with CH₂Cl₂/EtOAc, followed byrecrystallisation from CH₂Cl₂/hexane (below 40° C.) to givedi-tert-butyl2-[[2-[bis(2-bromoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl phosphate(Ib-7E)(2.59 g, 72%) as an unstable yellow solid: mp 99-101° C. (dec);¹H NMR [(CD₃)₂SO] δ 8.93 (t, J=5.6 Hz, 1 H), 8.76 (d, J=2.8 Hz, 1 H),8.33 (d, J=2.8 Hz, 1 H), 4.01 (g, J=6.1 Hz, 2 H), 3.62-3.42 (m, 10 H),1.43 (s, 18 H). HRMS (FAB) calcd for C₂₁H₃₄ ⁷⁹Br₂N₄O₉P (MH⁺) m/z675.0430 found 675.0398; calcd for C₂₁H₃₄ ⁷⁹Br⁸¹BrN₄O₉P (MH⁺) m/z677.0410, found 677.0397; calcd for C₂₁H₃₄ ⁸¹Br₂N₄O₉P (MH⁺) m/z679.0389, found 679.0398. Anal. (C₂₁H₃₃Br₂N₄O₉P).

A solution of Ib-7E (2.80 g, 4.14 mmol) and TFA (15 mL) in dry CH₂Cl₂(15 mL) was stirred at 20° C. for 1 h, then concentrated under reducedpressure. Residual TFA was removed azeotropically with CH₃CN (2×) andthe resulting residue was dissolved in EtOAc. Addition of excess hexaneprecipitated a semisolid which was dried under high vacuum at 20° C. togive Ib-7P (98%) as a yellow foam. ¹H NMR [(CD₃)₂SO] δ 8.93 (t, J=5.6Hz, 1 H), 8.75 (d, J=2.8 Hz, 1 H), 8.36 (d, J=2.8 Hz, 1 H), 3.97 (q,J=6.3 Hz, 2 H), 3.62-3.43 (m, 10 H). HRMS (FAB) calcd for C₁₃H¹⁸⁷⁹Br₂N₄O₉P (MH⁺) m/z 562.9178, found 562.9171; calcd for C₁₃H₁₈⁷⁹Br⁸¹BrN₄O₉P (MH⁺) m/z 564.9158, found 564.9152; calcd for C₁₃H₁₈⁸¹Br₂N₄O₉P. (MH⁺) m/z 566.9137, found 566.9121. Treatment of diacidIb-7P with NaHCO₃ (2.0 equiv.) gave the disodium salt.

Example 273-[[5-[Bis(2-Chloroethyl)Amino]-2,4-Dinitrobenzoyl]Amino]PropylDihydrogen Phosphate (Ia-3P)

Similar phosphorylation of IIa-3, followed by chromatography of theproduct on silica gel and elution with CH₂Cl₂/EtOAc (2:3), gavedi-tert-butyl3-[[5-[bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl]amino]propylphosphate (Ia-3E) (76%) as a yellow solid: mp (EtOAc/i-Pr₂O/hexane)120-121° C. (dec); ¹H NMR [(CD₃)₂SO] δ 8.70 (t, J=5.6 Hz, 1 H), 8.55 (s,1 H), 7.45 (s, 1 H), 3.96 (q, J=6.7 Hz, 2 H), 3.82 (t, J=5.8 Hz, 4 H),3.69 (t, J=5.8 Hz, 4 H), 3.34 (after D₂O exchange, t, J=6.8 Hz, 2 H),1.86 (pent, J =6.6 Hz, 2 H), 1.42 (s, 18 H). Anal. (C₂₂H₃₅Cl₂N₄O₉P) C,H, N.

Similar treatment of ester Ia-3E with TFA gave diacid Ia-3P (99%) as ahygroscopic yellow solid. ¹H NMR [(CD₃)₂SO] δ 8.71 (t, J=5.6 Hz, 1 H),8.54 (s, 1 H), 7.45 (s, 1 H), 3.92 (q, J=6.7 Hz, 2 H), 3.82 (t, J=5.8Hz, 4 H), 3.69 (t, J=5.8 Hz, 4 H), 3.31 (q, J=6.5 Hz, 2 H), 1.84 (pent,J=6.6 Hz, 2 H). HRMS (FAB) Calcd. for C₁₄H₂₀ ³⁵Cl₂N₄O₉P [M+H]⁺ m/z489.0345; found 489.0344. Calcd. for C₁₄H₂₀ ³⁵Cl³⁷ClN₄O₉P [M+H]⁺ m/z491.0316; found 491.0317. Calcd. for C₁₄H₂₀ ³⁷Cl₂N₄O₉P [M+H]⁺ m/z493.0286; found 493.0312. Treatment of diacid I-3P with NaHCO₃ (2:0equiv) gave the disodium salt.

Example 283-[[5-[Bis(2-Bromoethyl)Amino]-2,4-Dinitrobenzoyl]Amino]PropylDihydrogen Phosphate (Ia-8P)

Similar phosphorylation of IIa-8, followed by chromatography of theproduct on silica gel and elution with CH₂Cl₂/EtOAc (1:1), gavedi-tert-butyl3-[[5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyl phosphate(Ia-8E) (66%) as a yellow solid: mp (EtOAc/i-Pr₂O/hexane) 110-111° C.(dec). ¹H NMR ((CD₃)₂SO) δ 8.70 (t, J=5.6 Hz, 1 H), 8.55 (s, 1 H), 7.44(s, 1 H), 3.96 (q, J=6.7 Hz, 2 H), 3.79-3.63 (m, 84 H), 3.35 (after D₂Oexchange, t, J=6.8 Hz, 2 H), 1.86 (pent, J=6.6 Hz, 2 H), 1.42 (s, 18 H).Anal. (C₂₂H₃₅Br₂N₄O₉P) C, H, N.

Similar treatment of ester Ia-8E with TFA gave diacid Ia-8P (99%) as ahygroscopic yellow solid. ¹H NMR [(CD₃)₂SO] δ 8.71 (t, J=5.6 Hz, 1 H),8.55 (s, 1 H), 7.43 (s, 1 H), 3.93 (q, J=6.7 Hz, 2 H), 3.79-3.63 (m, 8H), 3.31 (q, J=6.5 Hz, 2 H), 1.85 (pent, J=6.6 Hz, 2 H). HRMS (FAB)calcd for C₁₄H₂₀ ⁷⁹Br₂N₄O₉P (MH⁺) m/z 576.9335, found 576.9314; calcdfor C₁₄H₂₀ ⁷⁹Br⁸¹BrN₄O₉P (MH⁺) m/z 578.9314, found 578.9305; calcd forC₁₄H₂₀ ⁸¹ Br₂N₄O₉P (MH⁺) m/z 580.9294, found 580.9297. Treatment ofdiacid Ia-8P with NaHCO₃ (2.0 equiv.) gave the disodium salt.

Example 292-[[2-[Bis(2-Chloroethyl)Amino]-3,5-Dinitrobenzoyl]Amino]EthylDihydrogen Phosphate (Ib-2P)

Similar phosphorylation of IIb-2, followed by chromatography of theproduct on silica gel and elution with CH₂Cl₂/EtOAc (13:7), gavedi-tert-butyl2-[[2-[bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl phosphate(Ib-2E) (72%) as a yellow solid: mp (EtOAc/i-Pr₂O/hexane) 107-108° C.(dec); ¹H NMR [(CD₃)₂SO] δ 8.90 (t, J=5.6 Hz, 1 H), 8.75 (d, J=2.8 Hz, 1H), 8.33 (d, J=2.8 Hz, 1 H), 4.01 (q, J=6.1 Hz, 2 H), 3.72 (t, J=6.8 Hz,4 H), 3.53 (q, J=5.5 Hz, 2 H), 3.43 (t, J=6.8 Hz, 4 H), 1.43 (s, 18 H).Anal. (C₂₁H₃₃Cl₂N₄O₉P) C, H, N, P. CRL 11363.

Similar treatment of ester Ib-2E with TFA gave diacid Ib-2P (98%) as ayellow foam. ¹H NMR [(CD₃)₂SO] δ 8.89 (t, J=5.6 Hz, 1 H), 8.74 (d, J=2.8Hz, 1 H), 8.36 (d, J=2.8 Hz, 1 H), 3.98 (q, J=6.2 Hz, 2 H), 3.72 (t,J=6.7 Hz, 4 H), 3.51 (q, J=5.6 Hz, 2 H), 3.43 (t, J=6.7 Hz, 4 H). HRMS(FAB) Calcd. for C₁₃H₁₈ ³⁵Cl₂N₂O₉P [M+H]⁺ m/z 475.0189; found 475.0189.Calcd. for C₁₃H₁₈ ³⁵Cl³⁷ClN₂O₉P [M+H]⁺ m/z 477.0159; found 477.0167.Calcd. for C₁₃H₁₈ ³⁵Cl₂N₂O₉P [M+H]⁺ m/z 479.0130; found 479.0160.Treatment of diacid Ib-2P with NaHCO₃ (1.0 equiv.) gave the monosodiumsalt.

Example 302-[(2-Chloroethyl)-2,4-Dinitro-6-[[[2-(Phosphonooxy)Ethyl]amino]-Carbonyl]Anilino]EthylMethanesulfonate (Ib-2mP)

Similar phosphorylation of IIb-2m, followed by chromatography of theproduct on silica gel and elution with EtOAc, gave2-[(2-chloroethyl)-2-(6-tert-butoxy-8,8-dimethyl-6-oxido-5,7-dioxa-2-aza-6-phosphanon-1-anoyl)-4,6-dinitroanilino]ethylmethanesulfonate (Ib-2mE) (80%) as a yellow foam. ¹H NMR [(CD₃)₂SO] δ8.94 (t, J=5.6 Hz, 1 H), 8.75 (d, J=2.8 Hz, 1 H), 8.34 (d, J=2.8 Hz, 1H), 4.28 (t, J=5.4 Hz, 2 H), 4.02 (q, j=6.2 Hz, 2 H), 3.74-3.43 (m, 8H), 3.13 (s, 3 H), 1.43 (s, 18 H). ¹³C NMR δ 265.6, 146.2, 145.3, 140.8,135.6, 127.5, 122.4, 81.7, 67.5, 64.2, 54.3, 51.3, 41.4, 36.5, 29.5.

Similar treatment of ester Ib-2mE with TFA gave diacid Ib-2mP (68%) as ayellow solid. Mp (EtOAc/CH₂Cl₂): 132-134° C.; ¹H NMR [(CD₃)₂SO] δ 8.92(t, J=5.6 Hz, 1 H), 8.74 (d, J=2.8 Hz, 1 H), 8.37 (d, J=2.8 Hz, 1 H),4.29 (t, J=5.4 Hz, 2 H), 3.98 (q, J=6.0 Hz, 2 H), 3.58-3.40 (after D₂Oexchange, m, 8 H), 3.13 (s, 2 H). ¹³C NMR δ 165.5, 146.1, 145.3, 140.8,135.7, 127.6, 122.3, 67.5, 63.3, 63.2, 54.3, 51.3, 41.3, 36.5. Anal.(C₁₄H₂₀ClN₄O₁₂PS) C, H, N.

Example 312-({2-[Bis(2-Bromopropyl)Amino]-3,5-Dinitrobenzoyl}amino)EthylDihydrogen Phosphate (Ib-7aP)

Similar phosphorylation of alcohol IIb-7a (0.67 g, 1.3 mmol) withdi-tert-butyl diethylphosphoramidite (93%, 489 mg, 2.0 mmol), followedby flash column chromatography on silica gel, eluting withEtOAc/petroleum ether (1:1) gave Ib-7aE as a yellow solid (0.74 g, 81%):mp (EtOAc/petroleum ether) 121-123° C.; ¹H NMR [(CD₃)₂SO] δ 9.09 (m, 1H), 8.73 (m, 1 H), 8.32 (m, 1 H), 4.44 (m, 2 H), 4.00 (m, 2 H), 3.39 (m,2 H), 3.60 (m, 4 H), 1.62 (m, 6 H), 1.44 (s, 18 H). ¹³C NMR δ 165.9,144.8, 143.6, 139.6, 133.2, 128.0, 123.1, 81.6, 64.0, 60.4, 39.9, 29.4,23.5. Anal. (C₂₃H₃₇Br₂N₄O₉P) C, H, N.

Similar treatment of Ib-7aE (100 mg) with TFA (6 mL), followed bycrystallization from CH₂Cl₂/EtOAc, gave Ib-7aP as a yellow solid (70 mg,85%): mp 157-161° C.; ¹H NMR [(CD₃)₂SO] δ 9.07 (m, 1 H), 8.72 (m, 1 H),8.36 (m, 1 H), 4.43 (m, 2 H), 4.00 (m, 2 H), 3.52 (m, 6 H), 1.62 (m, 6H). ¹³C NMR δ 165.9, 144.8, 143.6, 139.7, 133.4, 128.1, 123.1, 63.2,60.4, 47.9, 39.9, 23.5. Anal. (C₁₅H₂₁Br₂N₄O₉P) C, H, N.

Example 322-[(2-Bromoethyl)-2,4-Dinitro-6-[[[2-(Phosphonooxy)Ethyl]Amino]-Carbonyl]Anilino]EthylMethanesulfonate (Ib-12P)

Similar phosphorylation of IIa-12, followed by chromatography of theproduct on silica gel and elution with EtOAc, gave2-[(2-bromoethyl)-2-(6-tert-butoxy-8,8-dimethyl-6-oxido-5,7-dioxa-2-aza-6-phosphanon-1-anoyl)-4,6-dinitroanilino]ethylmethanesulfonate (Ib-12E) (66%) as a yellow foam. ¹H NMR [(CD₃)₂SO] δ8.94 (t, J=5.6 Hz, 1 H), 8.75 (d, J=2.8 Hz, 1 H), 8.34 (d, J=2.8 Hz, 1H), 4.28 (t, J=5.4 Hz, 2 H), 4.02 (q, j=6.2 Hz, 2 H), 3.62-3.43 (m, 8H), 3.13 (s, 3 H), 1.43 (s, 18 H). HRMS (FAB) calcd for C₂₂H₃₇⁷⁹BrN₄O₁₂PS [M+H]⁺ m/z 693.1029; found 693.1010.

Similar treatment of ester Ib-12E with TFA gave diacid Ib-12P (98%) as ayellow foam. ¹H NMR [(CD₃)₂SO] δ 8.92 (t, J=5.6 Hz, 1 H), 8.74 (d, J=2.8Hz, 1 H), 8.37 (d, J=2.8 Hz, 1 H), 4.28 (t, J=5.4 Hz, 2 H), 3.98 (q,J=6.0 Hz, 2 H), 3.58-3.40 (after D₂O exchange, m, 8 H), 3.13 (s, 2 H).HRMS (FAB) calcd for C₁₄H₂₁ ⁷⁹BrN₄O₁₂PS [M+H]⁺m/z 578.9798; found578.9784; calcd for C₁₄H₂₁ ⁸¹Br⁸¹BrN₄O₁₂PS [M+H]⁺m/z 580.9777; found580.9784. Treatment of diacid Ib-12P with NaHCO₃ (1.0 equiv) gave themonosodium salt.

Example 33 2-[[2-[Bis(2-Iodoethyl)Amino]-3,5-Dinitrobenzoyl]Amino]EthylDihydrogen Phosphate (Ib-14P)

Similar phosphorylation of Ib-14, followed by chromatography of theproduct on silica gel and elution with CH₂Cl₂/EtOAc (3:1), gavedi-tert-butyl2-[[2-[bis(2-iodoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl phosphate(Ib-14E) (67%) as a yellow solid: mp (CH₂Cl₂/i-Pr₂O/hexane) 108-110° C.(dec); ¹H NMR [(CD₃)₂SO] δ 8.91 (t, J=5.6 Hz, 1 H), 8.74 (d, J=2.8 Hz, 1H), 8.30 (d, J=2.8 Hz, 1 H), 4.01 (q, J=6.3 Hz, 2 H), 3.53 (q, J=5.7 Hz,2 H), 3.45 (t, J7.8 Hz, 4 H), 3.24 (after D₂O exchange, t, J=7.6 Hz, 4H), 1.44 (s, 18 H). Anal. (C₂₁H₃₃I₂N₄O₉P), C, H, N, P.

Similar treatment of ester Ib-14E with TFA gave diacid Ib-14P (97%) as ayellow foam. ¹H NMR [(CD₃)₂SO] δ 8.90 (t, J=5.6 Hz, 1 H), 8.73 (d, J=2.8Hz, 1 H), 8.34 (d, J=2.8 Hz, 1 H), 3.98 (q, J=6.4 Hz, 2 H), 3.49 (afterD₂O exchange t, J=5.6 Hz, 2 H), 3.45 (t, J=7.8 Hz, 4 H), 3.29 (t, J=7.7Hz, 4 H). HRMS (FAB) Calcd. for C₁₃H₁₈I₂N₄O₉ [M+H]⁺m/z 658.3911; found658.3907. Treatment of diacid Ib-14P with NaHCO₃ (2.0 equiv.) gave thedisodium salt.

Example 342-[(2-Iodoethyl)-2,4-Dinitro-6-({[2-(Phosphonooxy)Ethyl]Amino}Carbonyl)-Anilino]EthylMethanesulfonate (Ib-15P)

Similar phosphorylation of alcohol IIb-15 (1.68 g, 3.1 mmol) withdi-tert-butyl diethylphosphoramidite (93%, 1.15 g, 4.5 mmol), followedby flash column chromatography on silica gel, eluting withEtOAc/petroleum ether (1:1), and crystallization from EtOAc/petroleumether, gave Ib-15E as a yellow solid (2.23 g, 97%): mp (EtOAc/petroleumether) 109-111° C.; ¹H NMR [(CD₃)₂SO] δ 8.98 (m, 1 H), 8.76 (d, J=2.8Hz, 1 H), 8.33 (d, J=2.8 Hz, 1 H), 4.27 (m, 2 H), 4.00 (m, 2 H), 3.53(m, 2 H), 3.46 (m, 4 H), 3.14 (s, 3 H), 1.43 (s, 18 H). ¹³C NMR δ 165.5,145.6, 145.2, 140.8, 135.6, 127.4, 122.4, 81.7, 67.5, 64.2, 55.4, 50.7,39.9, 36.5, 29.3, 2.6. Anal. (C₂₂H₃₆IN₄O₁₂PS), C, H, N.

Similar treatment of Ib-15E (405 mg) with TFA (6 mL) and crystallizationof the product from CH₂Cl₂/petroleum ether gave diacid Ib-15P as ayellow solid (306 mg, 89%): mp 147-150° C.; ¹H NMR [(CD₃)₂SO] δ 8.93 (m,1 H), 8.74 (d, J=2.8 Hz, 1 H), 8.36 (d, J=2.8 Hz, 1 H), 4.27 (m, 2 H),4.00 (m, 2 H), 3.46 (m, 6 H), 3.31 (m, 2 H), 3.12 (s, 3 H). ¹³C NMR δ165.5, 145.6, 145.2, 140.8, 135.7, 127.6, 122.3, 67.6, 63.3, 55.5, 50.7,39.9, 36.5, 2.7. Anal. (C₁₄H₂₀IN₄O₉PS), C, H, N.

Example 352-[(2-Chloroethyl)-2,4-Dinitro-3-[[[3-(Phosphonooxy)Propyl]Amino]-Carbonyl]Anilino]EthylMethanesulfonate (Ic-6P)

Similar phosphorylation of IIc-6, followed by chromatography of theproduct on silica gel and elution with EtOAc/petroleum ether (from 1:2to 1:1), gave2-[(2-chloroethyl)-3-(7-tert-butoxy-9,9-dimethyl-7-oxido-6,8-dioxa-2-aza-7-phosphahex-1-anoyl)-2,4-dinitroanilino]ethylmethanesulfonate (Ic-6E) (98%) as a yellow solid: mp (EtOAc/petroleumether) 98-102° C. ¹H NMR [(CD₃)₂SO] δ 8.83 (t, J=5.6 Hz, 1 H), 8.26 (d,J=9.4 Hz, 1 H), 7.65 (d, J=9.4 Hz, 1 H), 4.29 (t, J=5.3 Hz, 2 H), 3.92(q, J=6.7 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.62-3.55 (m, 2 H), 3.23 (q,J=6.5 Hz, 2 H), 3.15 (s, 3 H), 1.79 (pent, J=6.7 Hz, 2 H), 1.42 (s, 18H). ¹³C NMR δ 161.3, 146.4, 140.4, 137.6, 129.1, 127.6, 123.0, 81.2,66.8, 64.1, 64.0, 52.9, 50.1, 41.4, 36.6, 35.9, 29.3. Anal.(C₂₃H₃₈ClN₄O₁₂PS) C, H, N.

Similar treatment of ester Ic-6E with TFA gave diacid Ic-6P (84%) as ayellow solid: mp (EtOAc/CH₂Cl₂) 98-102° C.; ¹H NMR [(CD₃)₂SO] δ 8.84 (t,J=5.7 Hz, 1 H), 8.26 (d, J=9.4 Hz, 1 H), 7.65 (d, J=9.4 Hz, 1 H), 4.28(t, J=5.3 Hz, 2 H), 3.88 (q, J=6.8 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.53(after D₂O exchange, t, J=6.0 Hz, 2 H), 3.23 (q, J=6.6 Hz, 2 H), 3.15(s, 3 H), 1.76 (pent, J=6.7 Hz, 2 H). Anal. (C₁₅H₂₂ClN₄O₁₂PS) C, H, N.

Example 363-({3-[Bis(2-Bromoethyl)Amino]-2,6-Dinitrobenzoyl}Amino)PropylDihydrogen Phosphate (Ic-8P)

Similar phosphorylation of alcohol IIc-8 (1.41 g, 2.83 mmol) withdi-tert-butyl diethylphosphoramidite (93%, 1.25 g, 5.0 mmol), followedby flash column chromatography on silica gel, eluting withEtOAc/petroleum ether (1:1), gave Ic-8E as a yellow solid (1.77 g, 91%):mp (EtOAc/petroleum ether) 112-114° C.; ¹H NMR [(CD₃)₂SO] δ 8.86 (m, 1H), 8.24 (d, J=9.4 Hz, 1 H), 7.63 (d, J=9.4 Hz, 1 H), 3.92 (m, 2 H),3.70 (m, 4 H), 3.60 (m, 4 H), 3.22 (m, 2 H), 1.78 (m, 2 H), 1.41 (s, 18H). ¹³C NMR δ 161.4, 145.9, 139.9, 137.3, 129.2, 127.8, 122.5, 81.3,64.1, 52.5, 35.9, 30.1, 29.4. 29.1. Anal. (C₂₂H₃₅Br₂N₄O₉P), C, H, N.

Similar treatment of Ic-8E (900 mg) with TFA (10 mL) gave diacid Ic-8Pas a yellow foam (754 mg, 100%): ¹H NMR [(CD₃)₂SO] δ 8.83 (m, 1 H), 8.24(d, J=9.4 Hz, 1 H), 7.63 (d, J=9.4 Hz, 1 H), 3.86 (m, 2 H), 3.73 (m, 4H), 3.60 (m, 4 H), 3.22 (m, 2 H), 1.76 (m, 2 H). ¹³C NMR δ 161.3, 145.9,140.1, 137.4, 129.2, 127.6, 122.5, 62.9, 52.5, 36.0, 30.0, 29.3. HRMS(FAB) calcd for C₁₄H₂₀ ⁷⁹Br₂N₄O₉P. [M+H]⁺ m/z 576.9335, found 576.9326.

Example 372-[(2-Bromoethyl)-2,4-Dinitro-3-[[[2-(Phosphonooxy)Ethyl]Amino]-Carbonyl]Anilino]EthylMethanesulfonate (Ic-12P)

Similar phosphorylation of IIc-12, followed by chromatography of theproduct on silica gel and elution with EtOAc/petroleum ether (from 1:2to 1:0), gave (Ic-12E) (99%) as a yellow solid: mp (EtOAc/petroleumether) 82-86° C. (dec). ¹H NMR [(CD₃)₂SO] δ 9.00 (t, J=5.6 Hz, 1 H),8.26 (d, J=9.4 Hz, 1 H), 7.65 (d, J=9.4 Hz, 1 H), 4.28 (t, J=5.3 Hz, 2H), 3.92 (q, J=6.7 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.62-3.55 (m, 2 H),3.23 (q, J=6.5 Hz, 2 H), 3.15 (s, 3 H), 1.42 (s, 18 H). Anal.(C₂₂H₃₆BrN₄O₁₂PS) C, H, N.

Similar treatment of ester Ic-12E with TFA gave diacid Ic-12P (100%) asa yellow solid: mp (EtOAc/CH₂Cl₂) 93-97° C.; ¹H NMR [(CD₃)₂SO] δ 8.99(t, J=5.7 Hz, 1 H), 8.26 (d, J=9.4 Hz, 1 H), 7.65 (d, J=9.4 Hz, 1 H),4.28 (t, J=5.3 Hz, 2 H), 3.88 (q, J=6.8 Hz, 2 H), 3.72-3.62 (m, 4 H),3.53 (after D₂O exchange, t, J=6.0 Hz, 2 H), 3.23 (q, J=6.6 Hz, 2 H),3.15 (s, 3 H). Anal. (C₁₄H₂₀BrN₄O₁₂PS) C, H, N.

Example 382-[(2-Bromoethyl)-2,4-Dinitro-3-[[[3-(Phosphonooxy)Propyl]Amino]-Carbonyl]Anilino]EthylMethanesulfonate (Ic-13P)

Similar phosphorylation of IIc-13, followed by chromatography of theproduct on silica gel and elution with CH₂Cl₂/EtOAc (1:3), gave2-[(2-bromoethyl)-3-(7-tert-butoxy-9,9-dimethyl-7-oxido-6,8-dioxa-2-aza-7-phosphahex-1-anoyl)-2,4-dinitroanilino]ethyl methanesulfonate (Ic-13E) (70%) as ayellow solid: mp (CH₂Cl₂/i-Pr₂O) 95-96° C. (dec). ¹H NMR [(CD₃)₂SO] δ8.83 (t, J=5.6 Hz, 1 H), 8.26 (d, J=9.4 Hz, 1 H), 7.65 (d, J=9.4 Hz, 1H), 4.28 (t, J=5.3 Hz, 2 H), 3.92 (q, J=6.7 Hz, 2 H), 3.72-3.62 (m, 4H), 3.62-3.55 (m, 2 H), 3.23 (q, J=6.5 Hz, 2 H), 3.15 (s, 3 H), 1.79(pent, J=6.7 Hz, 2 H), 1.42 (s, 18 H). Anal. (C₂₃H₃₈BrN₄O₁₂PS) C, H, N,P.

Similar treatment of ester Ic-13E with TFA gave diacid Ic-13P (98%) as ahygroscopic yellow solid. ¹H NMR [(CD₃)₂SO] δ 8.84 (t, J=5.7 Hz, 1 H),8.26 (d, J=9.4 Hz, 1 H), 7.65 (d, J=9.4 Hz, 1 H), 4.28 (t, J=5.3 Hz, 2H), 3.88 (q, J=6.8 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.53 (after D₂Oexchange, t, J=6.0 Hz, 2 H), 3.23 (q, J=6.6 Hz, 2 H), 3.15 (s, 3 H),1.76 (pent, J=6.7 Hz, 2 H). HRMS (FAB) calcd for C₁₅H₂₃ ⁷⁹BrN₄O₁₂PS[M+H]⁺ m/z 592.9954; found 592.9956. Treatment of diacid Ic-13P withNaHCO₃ (1:0 equiv) gave the monosodium salt.

Example 392-[(2-Iodoethyl)-2,4-Dinitro-3-[[[3-(Phosphonooxy)Propyl]Amino]-Carbonyl]Anilino]EthylMethanesulfonate (Ic-15P)

Similar phosphorylation of IIc-15, followed by chromatography of theproduct on silica gel and elution with CH₂Cl₂/EtOAc (1:3), gave2-[(2-iodoethyl)-3-(7-tert-butoxy-9,9-dimethyl-7-oxido-6,8-dioxa-2-aza-7-phosphahex-1-anoyl)-2,4-dinitroanilino]ethylmethanesulfonate (Ic-15E) (58%) as a yellow solid: mp (EtOAc/iPr₂O)90-100° C. ¹H NMR [(CD₃)₂SO] δ 8.86 (t, J=5.6 Hz, 1 H), 8.25 (d, J=9.4Hz, 1 H), 7.63 (d, J=9.5 Hz, 1 H), 4.27 (t, J=5.2 Hz, 2 H), 3.91 (q,J=6.7 Hz, 2 H), 3.67 (t, J=5.2 Hz, 2 H), 3.60 (t, J=7.1 Hz, 2 H),3.26-3.17 (after D₂O exchange, m, partially obscured, 2 H), 3.23 (q,J=6.5 Hz, 2 H), 3.15 (s, 3 H), 1.78 (pent, J=6.6 Hz, 2 H). Anal.(C₂₃H₃₈IN₄O₁₂PS) C, H, N, P.

Similar treatment of ester Ic-15E with TFA gave diacid Ic-15P (97%) as ahygroscopic yellow solid: mp (CH₃CN/EtOAc) 84-86° C. ¹H NMR [(CD₃)₂SO] δ8.90 (t, J=5.6 Hz, 1 H), 8.24 (d, J=9.4 Hz, 1 H), 7.57 (d, J=9.5 Hz, 1H), 4.25 (t, J=5.2 Hz, 2 H), 3.81 (after D₂O exchange, q, J=6.7 Hz, 2H), 3.62 (after D₂O exchange, t, J=5.2 Hz, 2 H), 3.56 (t, J=7.1 Hz, 2H), 3.26 (t, J=6.9 Hz, 2 H), 3.20 (q, J=6.5 Hz, 2 H), 3.09 (s, 3 H),1.73 (pent, J=6.6 Hz, 2 H). HRMS(FAB) calcd for C₁₅H₂₂IN₄O₁₂PS (MH⁺) m/z640.9816. Found; 640.9795. Anal. (C₁₅H₂₂IN₄O₁₂PS) C, H.

TABLE 2 Combustion analysis data for new compounds of Tables 1a and 1bFound Calculated No C H N other C H N other IIa-1 44.5 3.9 18.6 44.6 4.118.9 IIa-3 41.3 4.3 13.7 17.4 (Cl) 41.1 4.4 13.7 17.3 (Cl) IIa-7 32.63.3 11.6 33.3 (Br) 32.3 3.3 11.6 33.0 (Br) IIa-7s 33.4 3.7 7.8 32.5 3.78.1 IIa-8 33.9 3.6 11.4 32.1 (Br) 33.8 3.6 11.3 32.1 (Br) IIa-9 35.5 3.810.7 31.2 (Br) 35.2 3.9 10.9 31.2 (Br) IIa-14 27.3 2.6 9.6 43.8 (I) 27.02.8 9.7 43.9 (I) IIb-1 51.2 5.7 15.9 51.3 5.7 15.9 IIb-3 41.6 4.5 13.617.1 (Cl) 41.1 4.4 13.7 17.3 (Cl) IIb-7 32.9 3.3 11.5 33.3 (Br) 32.3 3.311.6 33.0 (Br) IIb-7a 35.3 3.8 10.9 35.2 3.9 10.9 IIb-8 34.9 3.7 11.332.3 (Br) 33.8 3.6 11.3 33.3 (Br) IIb-14 27.8 3.1 9.5 27.0 2.8 9.7IIc-12 33.8 3.7 11.0 33.7 3.8 11.2 IIc-13 35.4 3.9 11.0 35.2 4.1 10.9IIc-14 36.7 4.5 10.2 36.4 4.4 10.6 Ib-7E 37.7 4.9 8.3  4.6 (P) 37.3 4.98.3  4.6 (P) Ib-2E 44.8 6.2 9.0  5.1 (P) Ib-14E 32.9 4.2 7.2  3.8 (P)32.7 4.3 7.3  4.0 (P) Ia-3E 44.2 5.9 9.3 43.9 5.9 9.3 Ia-8E 38.5 5.0 8.238.3 5.1 8.1 Ic-13E 39.0 5.4 8.9  4.4 (P) 39.2 5.4 7.9  4.4 (P) Ic-15E37.0 5.0 7.3  4.2 (P) 36.7 5.1 7.5  4.1 (P) Ic-15P 28.1 3.5 28.3 3.5

Representative alcohols of Formula (I) (listed in Table 1a) showselective cytotoxicity towards human cancer cell lines transfected witheither the E. coli nitroreductase cDNA (NTR) (Table 3, columns 2 and 3),or human cytochrome P450 reductase (P450R) under hypoxic conditions(Table 3, columns 4 and 5). In this table, sensitivity ratios aredisplayed to indicate the degree of selectivity for either NTRexpression (column 3) or hypoxia (column 5). However, overexpression ofP450R is not required for hypoxic selectivity.

IC₅₀ values are derived from cell proliferation experiments, following 4hour drug exposure under a gas phase of either 20% oxygen or 0% oxygen(anoxia, achieved using an anaerobic chamber). Cells were grown underaerobic conditions for a further 5 days, and cell density estimatedusing the sulphorhodamine B assay, to determine the concentration ofprodrug required to inhibit growth to 50% of control.

TABLE 3 Selective cytotoxicities of representative examples of thealcohols of Table 1a Human lung (4 h) A549 Human colon (4 h) A549(P450R^(+ve)) WiDr WiDr (P450R^(+ve)) 20% (NTR^(+ve)) WT:NTR anoxiaO₂/anoxia No IC₅₀ (μM) IC₅₀ Ratio IC₅₀ (μM) IC₅₀ Ratio  IIa-1 5.2 34 3.728 IIa-2 48 26 25 3.7 IIa-3 47 36 54 23 IIa-7 1.5 99 6.7 49 IIa-7s 9.335 2.1 109 IIa-8 1.6 224 23 6.6 IIa-9 6.4 58 22 9.4 IIa-10 10 22 — —IIa-11 11 9 — — IIa-12 4.2 116 73 10 IIa-13 5 90 32 18 IIa-14 2.9 49 134.5 IIb-1 61 2 384 <1.3 IIb-2 11.8 47 18 20 IIb-3 13.6 59 30 9 IIb-4 1418 — — IIb-5 13 19 — — IIb-6 27 5 — — IIb-7 0.3 61 0.8 56 IIb-7a 0.5 271.0 5.3 IIb-8 0.4 13 1.1 24 IIb-9 0.9 5 1.4 20 IIb-10 0.9 2 2.3 11IIb-11 1.0 2 6.6 4.5 IIb-12 0.4 48 0.28 133 IIb-13 0.3 27 0.15 138IIb-14 0.8 12 1.0 27 IIb-15 0.3 31 0.28 118 IIc-7 10 46 3.9 40 IIc-8 5.070 6.6 24 IIc-9 31 6 7.3 21 IIc-12 5.0 84 2.6 173 IIc-13 4.3 95 4.5 134IIc-14 20 16 7.1 57

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a depicts in vivo activity of compound IIb-7 and itscorresponding phosphate pre-prodrug Ib-7P, relative to the known hypoxiccytotoxin tirapazamine;

FIG. 1 b depicts an excision assay method: 300-500 mg Rif-1 xenograft→15 Gy→ Single prodrug dose @ MTD→ 18 hr→ excision→ disaggregate→ plate10²-10⁵ cells→ 12 days→ colonies counted;

FIG. 2 a depicts in vivo activity of compound IIb-7 and itscorresponding phosphate pre-prodrug, Ib-7P, against human coloncarcinoma xenografts grown by inoculating mixtures of 90% NTR−ve and 10%NTR+ve Wi Dr cells;

FIG. 2 b are plots of % animals survival versus time (days), in whichopen circles denote wild-type WiDR human colon carcinoma xenografts, andfilled circles denote tumours containing WiDr cells transfected with theE. coli NTR gene;

FIG. 3 a is a plot of plasma concentration versus time (min) forcompound Ib-2mP in female CD-1 mice;

FIG. 3 b is a plot of plasma concentration versus time (min) forcompound Ib-7P in female CD-1 mice;

FIG. 3 c is a plot of plasma concentration versus time (min) forcompound Ib-12P in female CD-1 mice;

FIG. 3 d is a plot of plasma concentration versus time (min) forcompound Ic-12P in female CD-1 mice.

The activity of the phosphates as hypoxic cytotoxins is demonstrated bythe data in FIG. 1 for the representative example (Ib-7P). This employsan in vivo excision assay with the Rif-1 tumour, where the oxic tumourcells are sterilised using 15 Gy of radiation, and the cytotoxicity ofan agent against the remaining hypoxic cells can be quantitated.Unexpectedly, the activity of the phosphate Ib-7P is found to exceedthat of its parent alcohol (IIb-7) at their respective maximum tolerateddoses (Ib-7P=750 μmol/kg; IIb-7=1000 μmol/kg). This experimentdemonstrates that the phosphate Ib-7P is more active against hypoxiccells than the reference hypoxic cytotoxin tirapazamine, and that it ismore active against hypoxic cells than oxic cells (i.e. when given postirradiation than when given without irradiation). Thus Ib-7P acts as ahypoxia-selective cytotoxin in vivo. Although less active against oxictumour cells, this activity is significant demonstrating that thecompound also has utility as a single agent (without radiation).

The notable activity of the phosphates of Formula (I) against hypoxiccells in human tumour xenografts is illustrated by the data of Table 4.In these experiments SiHa human cervical carcinoma cells were grownsubcutaneously in CD-1 nude (immunodeficient) mice. The compounds wereadministered at doses corresponding to 75% or 20% of the maximumtolerated dose (MTD), 5 minutes after a whole body dose of ionisingradiation (cobalt-60 gamma radiation) sufficient to sterilise the oxiccells (15 Gy). The tumours were excised 18 hours later, dissociated witha cocktail of proteases, and cell survival was determined using aclonogenic assay. The logarithms of cell kill were calculated from thedifference in the numbers of clonogens per gram tumour tissue betweentreated and control tumours. All of the phosphates tested showed largeeffects against hypoxic cells at 75% of the MTD (Table 4, column 4).This was selective for hypoxia as demonstrated by the lesser cell killin the absence of radiation. However, cell killing by the compoundsalone was significant in all cases (Table 4, column 5) demonstratingthat the compounds also have antitumour activity as single agents.Activity against hypoxic cells was also demonstrated for the samecompounds at doses corresponding to only 20% of the MTD (Table 4, column7). The reference hypoxic cytotoxin tirapazamine, and the referencenitrogen mustards (melphalan, chlorambucil and cyclophosphamide) lackedactivity at 20% of their respective MTDs.

TABLE 4 Activity of phosphates of Formula (I) against oxic and hypoxiccells in SiHa human tumour xenografts in nude mice. Compounds wereadministered as single i.p. doses in saline. 75% of MTD 20% of MTD Logkill, Log kill, Log kill, MTD Dose cmpd only^(a) cmpd after Dose cmpdafter No (μmol/kg) (μmol/kg) Mean ± sem radiation^(b) (μmol/kg)radiation^(b) Ia-8P 1000 750 ND^(c) ND 200 −0.01 ± 0.19  Ib-2P 237 178ND ND 47 −0.10 ± 0.08  Ib-2mP 1780 1330 2.47 ± 0.08 ≧3.47 ± 0.17 3562.56 ± 0.37 Ib-7P 750 562 1.20 ± 0.07    2.35 ± 0.17 150 1.12 ± 0.13Ib-12P 1330 1000 1.32 ± 0.30 ≧3.38 ± 0.28 267 1.65 ± 0.19 Ib-14P 562 4220.92 ± 0.06    1.93 ± 0.21 113 0.64 ± 0.11 Ib-15P 1330 1000 2.24 ± 0.15≧3.27 ± 0.13 267 1.74 ± 0.23 Ic-6P 3160 2370 1.11 ± 0.20 ≧2.62 ± 0.17632 1.64 ± 0.30 Ic-8P 562 422 ND ND 113 0.72 ± 0.08 Ic-12P 1780 13301.04 ± 0.29 ≧2.96 ± 0.31 356 2.03 ± 0.25 Ic-13P 3160 2370 2.36 ± 0.28≧3.23 ± 0.08 632 1.60 ± 0.20 Ic-15P 1780 1333 2.27 ± 0.17    2.80 ± 0.40356 1.81 ± 0.23 Tirapazamine 316 237 −0.02 ± 0.01     0.66 ± 0.11 63−0.01 ± 0.09  Chlorambucil 237 178 0.11 ± 0.04    0.31 ± 0.10 47 0.18 ±0.13 Melphalan 42.2 31.6 ND ND 8.4 0.04 ± 0.05 Cyclophosphamide 750 562ND ND 150 0.07 ± 0.10 ^(a)Relative to controls, in the same experiment,treated with vehicle (saline) only. Log kill = log₁₀ (clonogens/g tumourfor control tumours) − log₁₀(clonogens/g tumour for treated tumours).^(b)Relative to the mean for radiation only, in the same experiment. Logkill = log₁₀(clonogens/g tumour for radiation alone) − log₁₀(clonogens/gtumour for tumours treated with radiation plus compound). ^(c)Nonedetected

A representative example of the phosphates of Formula (I) as NTRactivated cytotoxins is provided in FIG. 2. In the WiDr in vivo growthdelay assay, xenografts containing mixtures of WiDr^(WT) and WiDr^(NTR)cells are grown to 300 mm³ and treated with a single dose of prodrug atits MTD. Tumour growth is monitored over time and animals are euthanasedwhen mean tumour volume >1600 mm³. Data is presented as time to death.Unexpectedly, the activity of the phosphate (Ib-7P) is observed toexceed that of its parent alcohol (IIb-7), when administered at theirrespective maximum tolerated doses (750 μmol/kg). Ib-7P is superior toIIb-7 with respect to (i) time to first treatment failure (77-days vs.17-days) and (ii) over-all survival (40% vs. 6%).

Pharmocokinetics of the phosphate esters Ib-2mP, Ib-7P, Ib-12P andIc-12P following administration to female CD-1 nude mice byintraperitoneal injection at a dose corresponding to 75% of the maximumtolerated dose. Monosodium salts of the compounds were dissolved inphosphate buffered saline, pH 7.4, with addition of one equivalent ofsodium bicarbonate. Serial blood samples were obtained by small tailvein bleeds, and 10 ul of plasma was prepared from each. Proteins wereprecipitated by addition of 3 volumes of methanol, and concentrations ofthe phosphate esters and corresponding alcohols were determined by HPLCusing either UV or mass spectrometry detection.

The data shows that the phosphate esters are converted efficiently tothe corresponding alcohols in mice. The alcohols are the speciesactivated by nitroreduction in hypoxic, or NTR-expressing, cells.

Wherein the foregoing description reference has been made to reagents,or integers having known equivalents thereof, then those equivalents areherein incorporated as if individually set forth.

While this invention has been described with reference to certainembodiments and examples, it is to be appreciated that furthermodifications and variations may be made to embodiments and exampleswithout departing from the scope of the invention.

1. A phosphate compound of Formula (I)

wherein: X represents at any available ring position —CONH—, —SO₂NH—,—O—, —CH₂, —NHCO— or —NHSO₂—; R represents a lower C₁₋₆ alkyl optionallysubstituted with one or more groups selected from hydroxy, amino andN-oxides therefrom or dialkylamino and N-oxides therefrom; Y representsat any available ring position —N-aziridinyl, —N(CH₂CH₂W)₂ or—N(CH₂CHMeW)₂, where each W is independently selected from halogen or—OSO₂Me; Z represents at any available ring position —NO₂, -halogen,—CN, —CF₃ or —SO₂Me; and pharmaceutically acceptable salts thereof.
 2. Aphosphate compound of Formula (I) as claimed in claim 1 which isselected from a compound represented by formulae (Ia), (Ib) or (Ic)

wherein Y represents

and wherein n represents1 to 6 Z represents —NO₂, -halogen, —CN, —CF₃ or—SO₂Me; and where each W is independently selected from halogen or—OSO₂Me and pharmaceutically acceptable salts thereof.
 3. The phosphatecompound of Formula (I) as claimed in claim 1 which is selected from:2-[[2-[Bis(2-bromoethyl)amino]-3,5-dinitrobenzoyl]amino] ethyldihydrogen phosphate;3-[[5-[Bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl]amino]propyldihydrogen phosphate;3-[[5-[Bis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyldihydrogen phosphate;2-[[2-[Bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyldihydrogen phosphate;2-[(2-Chloroethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino-carbonyl]anilino]ethylmethanesulfonate;2-({2-[Bis(2-bromopropyl)amino]-3,5-dinitrobenzoyl}amino)ethyldihydrogen phosphate;2-[(2-Bromoethyl)-2,4-dintro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;2-[[2-[Bis(2-iodoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogenphosphate;2-[(2-iodoethyl)-2,4-dinitro-6-({[2-phosphonooxy)ethyl]amino}carbonyl)-anilino]ethylmethanesulfonate;2-[(2-Chloroethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate;3-({3-[Bis(2-bromoethyl)amino]-2,6-dinitrobenzoyl}amino)propyldihydrogen phosphate;2-[(2-Bromoethyl)-2,4-dinitro-3-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;2-[(2-Bromoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate; and2-[(2-iodoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate.
 4. A method of preparing a phosphate represented bythe general formula (I);

wherein: X represents at any available ring position —CONH—, —SO₂NH—,—O—, —CH₂—, —NHCO— or —NHSO₂—; R represents a lower C₁₋₆ alkyloptionally substituted with one or more groups including hydroxy, aminoand N-oxides therefrom or dialkylamino and N-oxides therefrom; Yrepresents at any available ring position —N-aziridinyl or —N(CH₂CH₂W)₂,where each W is independently selected from halogen or —OSO₂Me; Zrepresents at any available ring position —NO₂, -halogen, —CN, —CF₃ or—SO₂Me; and pharmaceutically acceptable salts thereof: the methodcomprising the step of (i) phosphorylating a compound of formula (II)

wherein: X represents at any available ring position —CONH—, —SO₂NH—,—O—, —CH₂—, —NHCO— or —NHSO₂—; Y represents at any available ringposition —N-aziridinyl, —N(CH₂CH₂W)₂, or —N(CH₂CH MeW)₂ where each W isindependently selected from halogen or —OSO₂Me; Z represents at anyavailable ring position —NO₂, -halogen, —CN, —CF₃ or —SO₂Me; and Rrepresents a lower C₁₋₆ alkyl optionally substituted with one or moregroups including hydroxy, amino and N-oxides therefrom or dialkylaminoand N-oxides therefrom.
 5. A method of preparing a compound of formulae(Ia), (Ib) or (Ic)

wherein Y may represent

and wherein n represents1 to 6 Z represents —NO₂, -halogen, —CN, —CF₃ or—SO₂Me; and where each W is independently selected from halogen or—OSO₂Me and pharmaceutically acceptable salts thereof the methodcomprising the step of phosphorylating a compound represented byformulae (IIa), (IIb) or (IIc)

wherein Y represents

and wherein n represents1 to 6 Z represents —NO₂, -halogen, —CN, —CF₃ or—SO₂Me; and where each W is independently selected from halogen or—OSO₂Me and pharmaceutically acceptable salts.
 6. A method of killinghypoxic cells in a tumour comprising the step of administering an amountof a compound of Formula (I) as defined above in claim 1 to a subjectwith the tumour.
 7. A method of cell ablation utilising at least onenitroreductase enzyme comprising the step of using a compound of Formula(I) as defined above in claim 1 in an effective amount to ablate cellswhich express at least one nitroreductase enzyme.
 8. A method of cellablation utilising at least one nitroreductase enzyme comprising thestep of administering a compound of Formula (I) as defined above inclaim 1 in an effective amount to a subject to ablate cells whichexpress at least one nitroreductase enzyme.
 9. The method as claimed inclaim 8 wherein the at least one nitroreductase enzyme is encoded for bythe nfsB gene of either E. coli or by orthologous genes in Clostridiaspecies.
 10. The method as claimed in claim 8 wherein the cells thatexpress the at least one nitroreductase enzyme are tumour cells intissue in the subject.
 11. The method as claimed in claim 8 wherein thecell ablation is achieved through GDEPT (gene-directed enzyme-prodrugtherapy).
 12. The method as claimed in claim 8 wherein the cell ablationis achieved through ADEPT (antibody-directed enzyme-prodrug therapy).13. The method as claimed in claim 8 wherein the cells are mammalian.14. The method as claimed in claim 8 wherein the amount administered isbetween about 20% to 100% of the maximum tolerated dose of the subject.15. The method as claimed in claim 8 including the further step ofapplying irradiation or one or more chemotherapeutic agents to thesubject.
 16. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of Formula (I) as defined in claim 1 anda pharmaceutically acceptable excipient, adjuvant, carrier, buffer orstabiliser.
 17. A compound selected from:2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;2-[(2-Bromoethyl)-2,4-dinitro-3-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate;2-[(2-Bromoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate; and2-[(2-iodoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethylmethanesulfonate.
 18. The compound2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethylmethanesulfonate.
 19. A pharmaceutical composition comprising atherapeutically effective amount of a compound as claimed in claim 17 orclaim 18 and a pharmaceutically acceptable excipient, adjuvant, carrier,buffer or stabiliser.
 20. A method of killing hypoxic cells in a tumorcomprising the step of administering an effective amount of a compoundof claim 18 to a subject with a tumor.